EFFECT OF ENERGY DRINK CONSUMPTION ON

CARDIOVASCULAR RESPONSES DURING EXERCISE AND RECOVERY

A Thesis

Presented to the faculty of the Department of Kinesiology

California State University, Sacramento

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF SCIENCE

in

Kinesiology

(Exercise Science)

by

Brittany Elizabeth Joachim

FALL 2019

© 2019

Brittany Elizabeth Joachim

ALL RIGHTS RESERVED

ii EFFECT OF ENERGY DRINK CONSUMPTION ON

CARDIOVASCULAR RESPONSES DURING EXERCISE AND RECOVERY

A Thesis

by

Brittany Elizabeth Joachim

Approved by:

______, Committee Chair Roberto Quintana, PhD

______, Second Reader Troy Chinevere, PhD

______Date

iii

Student: Brittany Elizabeth Joachim

I certify that this student has met the requirements for format contained in the University format manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for the thesis.

______, Graduate Coordinator ______Katherine M. Jamieson, PhD Date

Department of Kinesiology

iv Abstract

of

EFFECT OF ENERGY DRINK CONSUMPTION ON

CARDIOVASCULAR RESPONSES DURING EXERCISE AND RECOVERY

by

Brittany Elizabeth Joachim

Statement of Problem

The purpose of this study was to determine if the consumption of a 24oz can of Monster

Energy® drink prior to exercise affected QTc during and after intense exercise.

Methodology

Fifteen subjects were recruited for the study on a volunteer basis. After initial informed consent and baseline measurements subjects visited the lab twice. The subjects were randomly assigned to consume 709 ml of Monster Energy® drink or 709 ml of a control drink with the same carbohydrate content as the energy drink. The subjects returned 4-14 days after the first trial to repeat the same procedures with the drink they hadn’t previously consumed.

After baseline ECG, BP, HR, body weight, and blood samples were taken, the subjects consumed either their assigned drink within a 30 min period. After 30 minutes, the subjects performed a maximal ergometer test. ECGs were continuously recorded during exercise.

Immediately upon exercise cessation, ECG, BP, HR were recorded and blood samples were taken. Subjects then laid supine for 10 minutes. After the 10 minutes, subjects were free to sit or stand two hours following exercise. During the 2-hour recovery period, ECG, BP, and HR were recorded at 1, 4, 7, and 10, 30, 60, 90 and 120 minutes following exercise.

v A two-factor repeated measures ANOVA was used to ascertain time x group interactions for QT/QTc interval, HR, BP, P wave and QRS complex durations. Significant main effects were analyzed by examining 95% confidence intervals. The significance level was set at α = 0.05 for all analyses.

Results

No significant difference was found in QTc between the ED and the placebo groups after the two randomized trials during rest and maximal exercise (p=0.05). However, QTc in placebo and ED group during max exercise was significantly decreased compared to any other time within each group (p<0.05). A significantly higher maximal power was achieved in the ED group vs. the placebo group (p<0.05). The ED group achieved a 4.2% higher mean max power output of

243.75 ± 92.22 W vs. 233.75 ± 90.62 W compared to the placebo group. There was significant

4.4% increase (p<0.05) time to maximal exercise exhaustion when receiving the ED compared to the placebo (0.4 minutes). Energy Drink consumption had no significant effect on SBP at any time interval compared to the placebo. Energy Drink consumption had no significant effect on

DBP at any time interval compared to the placebo. No statistically significant effect between ED consumption vs. placebo was found on heart rate. No effects were found between the ED and the placebo groups on RPE, as well.

Conclusions Reached

Our findings suggest that 709 mL ED consumption did not significantly affect the QTc interval at maximal exercise and but improved time to exhaustion by 0.4 minutes. Although ED consumption within 2 hours did not significantly affect HR and BP during and after a max exercise test in our study, further research might explore different volumes of EDs, different time parameters after the ED is consumed, and type of exercise after ED consumption on the

vi development of arrhythmias. A QTc change from baseline >60ms is expressed as a threshold for concern, and the QTc change from baseline in our study was within normal limits (44.8 ± 22.1 msec). Based on the limited results of this study, ED consumption is safe in healthy individuals

18-40 years old during and after exercise.

______, Committee Chair Roberto Quintana, PhD

______Date

vii

TABLE OF CONTENTS Page

List of Tables ...... x

List of Figures ...... xi

Chapter

1. INTRODUCTION ……………...………………………………………………………. 1

Purpose ...... 4

Significance ...... 4

Hypothesis ...... 4

Limitations ...... 5

Delimitations ...... 5

Assumptions...... 5

Definitions ...... 6

2. REVIEW OF LITERATURE ...... 7

Introduction ...... 7

Popularity of ED Consumption ...... 7

Adverse Events Related to ED Consumption ...... 8

Ingredients ...... 9

BP Responses after ED Consumption ...... 15

HR Responses after ED Consumption ...... 16

Basic Electrocardiogram (ECG) ...... 17

ECG Responses after ED Consumption ...... 18

QT Responses after ED Consumption ...... 19

viii Conclusion ...... 22

3. METHODS ...... 23

Study Design ...... 23

Study Participants ...... 23

Procedures ...... 24

Data Analysis ...... 27

4. RESULTS ...... 28

Study Participant Characteristics ...... 28

Systolic Blood Pressure ...... 29

Diastolic Blood Pressure ...... 30

Heart Rate ...... 31

Rate of Perceived Exertion ...... 32

Exercise Time ...... 33

Maximal Power ...... 34

Relative VO2 Max ...... 35

QT/QTc Interval ...... 36

5. DISCUSSION ...... 37

Conclusion ...... 42

References ...... 44

ix

LIST OF TABLES Tables Page

1. Summary of Procedures During each Experiment…….………………….…..…………26

2. Summary of Study Participant Characteristics………………….……………….………28

3. RPE Values presented as mean ± SD…………….……………………………….……. 32

x LIST OF FIGURES Figures Page

1. Pre-, max, and post-exercise systolic blood pressure following drink consumption……29

2. Pre-, max, and post-exercise diastolic blood pressure following drink consumption…...30

3. Pre-, max, and post-exercise heart rate following drink consumption…...………….…. 31

4. Time to exhaustion at maximal exercise between trials…………………..……………. 33

5. Maximal Power (W) in the ED and Placebo groups….……..……………………….…. 34

6. Relative VO2 (ml/kg/min) in the ED and Placebo groups ……………...………….….. 35

7. Pre-, max, and post-exercise QTc (msec) following drink consumption…..……….….. 36

xi 1

Chapter 1

Introduction

There are an abundance of energy drinks available on the market and their consumption has risen in popularity, particularly in younger populations, with a reported 42.3 percent of adolescents (11-18 yrs) consuming these beverages (Odea, 2003). In a national sample survey

(Mitchell, Knight, & Hockenberry, 2014), it was reported that 4 percent of individuals in the U.S. consumed at least one energy drink per day. Monster Energy® drink, first introduced in 2002, is one of the most popular energy drinks (Schmidt, McIntire, Caldwell, & Hallman, 2008).

However, the safety of consuming these popular beverages remains unclear. There were 5 deaths reported to the Food and Drug Administration (FDA) occurring after consumption of Monster

Energy® drinks from 2004-2012 (“CFSAN”, 2017). In an analysis of energy-drink toxicity reported in the National Poison Data from 2010-2011, major adverse effects included three cases of seizure, two cases of non-ventricular dysrhythmia, one case of ventricular dysrhythmia, and one case of tachypnea (Seifert, Seifert, & Schaechter, 2013). Based upon Mitchell et al. (2014) findings, it can be concluded that the incidence of an adverse event occurring in the U.S., in consumers of all ages who consume one energy drink per day is 0.00000132%. There was an additional lawsuit filed against Monster Energy® in June 2013 after the death of a teenager from cardiac arrest (Bronstad, 2013); the teenager had a habit of drinking two-16oz Monster Energy® drinks per day.

The main ingredient in energy drinks is caffeine. Caffeine consumption alone causes a

10mmHg rise in systolic blood pressure (SBP) in healthy study participants; however, an increased BP does not explain the development of arrhythmias possibly attributed to energy drinks (Steinke, Dhanapal, & Kalus, 2009). Additionally, caffeine intake alone is not associated with ECG irregularities, specifically QTc prolongation (QTc prolongation can be a marker of life 2 threatening cardiac arrhythmias) (Steinke et al., 2009). The proprietary “Energy Blend” synonymous with energy drinks may be the contributing factor in cardiac associated deaths after consumption of large volumes of energy drinks. The “Energy Blend” ingredients of Monster

Energy® drink may contribute to a change in BP or a change in heart function, and their exact interaction with each other is not clearly understood (Myers, 2003). The product packaging of an energy drink, 5-Hour ENERGY®, currently list a recommended maximum dose of two containers per day, and to be spaced several hours apart (5-hour ENERGY, 2017). However, Monster

Energy® drink product packaging does not contain any such warning/advisory about recommended dosing. 5-Hour ENERGY® contains 200mg of caffeine in two ounces (5-hour

ENERGY, 2017) while a 24oz can of and Monster Energy® drink contains 240mg of caffeine.

There have been a few studies examining the effect of energy drink (ED) consumption on electrocardiogram (ECG) rhythms. The effect of 5-hour ENERGY® on ECG and BP parameters showed no effect on the QT interval, but an approximate 5mmHg increase in SBP after 3 and 5 hours, and after 1 and 5 hours for Diastolic Blood Pressure (DBP) during rest (Shah, Dargush,

Potts, & Lee, 2016). While this study provided valuable information, it did not address the safety in study participants using higher amounts of energy drinks, a common practice in avid energy drink users (Toblin, Clarke-Walper, & Kok, 2012). A more recent study observed ECG responses over a 24-hour period after 18 study participants consumed two 16oz cans of Monster

Energy® drink during rest (Fletcher, Lacy, & Aaron, 2017). No changes in ECG patterns were observed at any time point, except a significant QTc prolongation of 10 ms was observed at 2 hours post-consumption (p=0.02). QTc prolongation greater than 60 ms from baseline is a recognized marker for increased risk for fatal arrhythmias (Fletcher et al., 2017). Another study examined the effect of ED consumption on ECG patterns during a 30-minute post-exercise period

(Wicklund, Karlsson, Ostrom, & Messner, 2009). With the exception of an increased PR interval 3 before exercise, no significant changes were observed in the PR, QRS, and QTc intervals. The absence of significant findings by Wicklund et al. (2009) might be due to insufficient time of monitoring during the post-exercise period, as was observed in the Fletcher et al. (2017) study where QTc was increased 2 hours after ED consumption.

A major reason that fitness enthusiasts/recreational athletes consume energy drinks is to enhance sport performance. Indeed, a recent unpublished survey conducted on 9,639 U.S. Air

Force members reported that 66% and 16% drink these beverages for increased stamina and endurance, respectively (Shah & Grayson, 2012). Although ED consumption prior to exercise appears to improve muscle strength and endurance performance, consuming energy drinks prior to exercise may raise the risk for a cardiac event (Souza et al., 2017). A review of published literature reported 17 cases of cardiovascular events or deaths were in part related to energy drinks (Goldfarb, Tellier, & Thanassoulis, 2014). Since cardiac arrhythmias and sudden cardiac death have been reported after high doses of energy drinks, a heightened level of awareness may be warranted about the safety of consuming energy drinks prior to vigorous exercise at least in those with occult or diagnosed heart disease (Kaski & Carro, 2013).

According to the product packaging, Monster Energy®, original flavor, 16oz can contains: Riboflavin (3.4mg), Niacin (40mg), Vitamin B6 (4mg), Vitamin B12 (12mcg), Taurine

(2000mg), Panax Ginseng (400mg), sugar (54g), sodium (360mg) and an ‘energy blend’

(5000mg) containing: L-carnitine, glucose, caffeine, guarana, inositol, glucuronolactone and maltodextrin (Monster Energy®”, 2013). Although not disclosed on the product packaging itself, independent labs state that Monster Energy® drink, original flavor, contains 160mg of caffeine per 16oz can (“Monster Energy®”, 2013). Since caffeine does not appear to alter the electrical activity of the heart it is possible that other ingredients contained in the ‘energy blend’ or 4 interactions of these ingredients may induce electrical disturbances of the heart, as well as an additional interaction with exercise.

Purpose

To date, limited data are available regarding the effect of consuming energy drinks on exercise and/post-exercise ECG responses in healthy study participants. Therefore, the purpose of this study is to examine exercise and post-exercise QTc responses after consuming an energy drink. A secondary purpose will be to compare ED versus control drink on maximal exercise performance.

Significance

To date, there is limited data on the effect of energy drink consumption on exercise and post-exercise ECGs in healthy study participants. With 17 cardiovascular adverse events

(Goldfarb et al., 2014) and 5 deaths reported after energy drink consumption (“CFSAN Adverse,”

2017) it is important to investigate the effect of energy drink consumption on ECG patterns.

With the increasing popularity of energy drinks, especially as an ergogenic aid, further research is needed to determine the effects of energy drink consumption prior to exercise on cardiovascular responses during and after exercise.

Hypotheses

1. There will be no significant difference in the QTc interval between the placebo-control and

Monster Energy® arms during or after maximal exercise test in young healthy volunteers

2. There will be no significant difference in SBP between the placebo-control and Monster

Energy® arms during or after maximal exercise test in young healthy volunteers

3. There will be no significant difference in DBP between the placebo-control and Monster

Energy® arms during or after maximal exercise test in young healthy volunteers 5

4. There will be no significant difference in HR between the placebo-control and Monster

Energy® arms during or after maximal exercise test in young healthy volunteers

5. There will be no significant difference in rate of perceived exertion between the placebo- control and Monster Energy® arms during the maximal exercise test in young healthy volunteers

6. There will be no significant difference in maximal exercise test time between the placebo- control and Monster Energy® arms during or after exercise in young healthy volunteers

7. There will be no significant difference in maximal exercise test power between the placebo- control and Monster Energy® arms during or after exercise in young healthy volunteers

8. There will be no significant difference in relative VO2 max between the placebo-control and

Monster Energy® arms during a maximal exercise test in young healthy volunteers

Limitations

In the current study, there is no discrimination between caffeine tolerant and caffeine naïve study participants. There is no control over the study participant’s diet regarding food consumption and habitual caffeine intake. During the trials, high-intensity exercise may interfere with the electrodes and ECG readings. An ergometer was used as the exercise modality to minimize this limitation. No generalizations can be made about the chronic effects of energy drink consumption and the effects of energy drink consumption on individuals under 18 and over 40.

Delimitations

Each study participant served as their own control. The study participants were 15-18 asymptomatic college students. The study participants were 18-40 years of age. The exercise test performed was a maximal VO2 max test rather than a steady state bout of exercise.

Assumptions

1. It was assumed that each study participant performed to his or her physiological limits during

each trial. 6

2. It was assumed that each study participant began each experimental trial in the same

physiological state.

3. It was assumed that all data collection and analysis equipment was properly calibrated and

maintained calibration throughout the trials.

Definitions

Electrocardiogram- (ECG) A recording of the electrical activity of the heart (Food and Drug

Administration, 2005)

Blood Pressure- (BP) The pressure, measured in millimeters of mercury, against the walls of the arteries (Chung, 2013)

Systolic blood pressure- (SBP) The maximum arterial pressure during contraction of the left ventricle of the heart, measured in millimeters of mercury (Chung, 2013)

Diastolic blood pressure- (DBP) The minimum arterial pressure during relaxation and dilation of the ventricles of the heart when the ventricles fill with blood, measured in millimeters of mercury

(Chung, 2013)

QT interval- (QTc) The duration of ventricular depolarization and subsequent repolarization

(Food and Drug Administration, 2005)

QT/QTc interval- QTc interval corrected so it is not a heart rate (HR) dependent value (Food and

Drug Administration, 2005)

Arrhythmia- a condition in which the heart beats with an irregular or abnormal rhythm (Goldfarb,

2014)

Cardiac arrest- a sudden, sometimes temporary, cessation of function of the heart (Goldfarb,

2014)

7

Chapter 2

Review of Literature

Introduction

In 1987, energy drinks (EDs) were first introduced in Austria (Ali, Rehman, Babayan,

Stapleton, & Joshi, 2015). Years later, a popular ED, Monster Energy®, was introduced in

Australia in 2002. Many EDs on the market today contain ingredients such as caffeine, taurine, and carbohydrates (Wicklund et al., 2009). Consumption of EDs has continuously increased over the past decade (Odea, 2003). In 2001, 22% of young college students reported energy drink consumption (Schmidt et al., 2003). This consumption continues to increase, especially in the adolescent population (Odea, 2003).

Popularity of ED Consumption

A study that interviewed 78 adolescents, evaluated their responses regarding supplement usage and their perceived benefits (Odea, 2003). Forty-two percent of these adolescents consumed an energy drink within two weeks prior to the experiment. The dramatic increase in energy drink consumption among adolescents may be because a large number of adolescents receive an insufficient amount of sleep, with only 20% of adolescents getting enough sleep daily

(Schmidt et al., 2003). In addition to receiving inadequate sleep, adolescents are awake at a later circadian cycle compared to older adults, which results in reduced alertness when they are awake

(Schmidt et al., 2003). The reasons for energy drink consumption given by adolescents in Odea

(2003) study were increased energy, taste, sports performance, soft drink substitute, peer group pressure, and attractive packaging. Actual responses from adolescents included “the energy gives you a lift”, “other guys on the team take it”, “coach makes us drink it”, and “there are a lot of vitamins in it” (Odea, 2003). The qualitative data from this study reflects only positive effects of energy drinks with no mention or concern for the potentially dangerous side effects. The author 8 also reported that peer pressure and pressure from coaches had an influence on the decision to consume energy drinks in adolescents.

When 400 U.S. Air Force members were surveyed, 61.01% of respondents reported energy drink usage, and 30.5% reported consuming energy drinks at least once a week (Schmidt et al., 2008). Monster Energy® drink and Red Bull Energy® drink were the participant’s primary choice of energy drink within the study. When participants were questioned as to their preference of energy drinks many participants responded, because of “health related” reasons as well as availability and price. Air Force members in this study reported that the number one energy drink effect was increased mental alertness (77.39%) followed by increased mental endurance

(39.13%). The negative effects of energy drink consumption were also recorded, with 31.3% of study participants reporting an increased HR, while 1.12% reported a heart arrhythmia (Schmidt,

2008). Concerns have arisen about a potential relationship between energy drinks consumption and adverse cardiovascular events (Grasser, Miles-Chan, Charriere, Loonam, & Dulloo, 2016).

There are limited data regarding the safety of energy drinks (Wicklund et al., 2009). Therefore, the relative safety of consuming these beverages remains unclear despite evidence that ED consumption may improve mental and physical performance.

Adverse Events Related to ED Consumption

Individuals consume EDs to enhance alertness and performance despite potential safety concerns involving ED consumption. There have been 17 cases of adverse cardiovascular events after the consumption of energy drinks including 5 atrial arrhythmias, 5 ventricular arrhythmias, 1

QT prolongation, and 4 ST segment elevations, and two cases of cardiac arrest (Goldfarb et al.,

2014). Of these 17 cases, only 1 person had previous cardiovascular disease. Out of the 11 cases with cardiac arrest, ventricular arrhythmia, or ST segment elevations, 5 people reported consuming acute heavy amounts of energy drinks. Four individuals of the 11 reported consuming 9 the energy drink with alcohol or other drugs (Goldfarb, 2014). Five deaths occurring after the consumption of Monster energy drinks have been reported to the FDA (“CFSAN Adverse,”

2017). The incidence of an adverse cardiovascular event after the consumption of an energy drink (Goldfarb et al., 2014) is 1: 6,347,647 (0.000016%).

Ingredients

The FDA markets energy drinks like Monster Energy® drink and Rockstar Energy® drink as dietary supplements (“CFSAN Adverse,” 2012). Due to adverse medical events, the

FDA warns individuals to consult their doctor before consuming any product labeled as an

“energy shot” or “energy drink” (CFSAN, 2012). Monster Energy®, original flavor, 16oz can contains: Riboflavin (3.4mg), Niacin (40mg), Vitamin B6 (4mg), Vitamin B12 (12mcg), Taurine

(2000mg), Panax Ginseng (400mg), sugar (54g), sodium (360mg) and an ‘energy blend’

(5000mg) containing: L-carnitine, glucose, caffeine, guarana, inositol, glucuronolactone and maltodextrin (Monster, 2013). Although not disclosed on the product packaging itself, independent labs state that Monster Energy® drink, original flavor, contains 160mg of caffeine per 16oz can (Monster, 2013). While a few brands of energy drinks are the most heavily consumed in the United States, there are over 200 varieties that contain many of the same ingredients (Grasser et al., 2016).

caffeine.

A main ingredient in energy drinks is caffeine. The effects of caffeine on the human body can be explained by three mechanisms of action: the antagonism of adenosine receptors, the mobilization of intracellular calcium, and the inhibition of phosphodiesterase. Caffeine’s primary mechanism of action is on the central nervous system. Caffeine blocks adenosine receptors, mainly types A1 and A2 in the central nervous system, and causes an increased release of dopamine, noradrenalin, and glutamate. High caffeine doses will induce adenosine antagonism 10

and inhibit phosphodiesterase, interacting with the sympathetic nervous system and inducing B1- receptor activation. This will result in an increase in HR.

Previously, the effects of caffeine were primarily attributed to promoting intracellular

Ca2+ or the inhibition of phosphodiesterase although these effects only occur with very high volumes of caffeine consumption (McLellan, Caldwell, & Lieberman, 2016). Consequently, it is now known that the central nervous system is the main mechanism of action involving caffeine that results from low to moderate doses. Specifically, low to moderate doses of caffeine block A1 and A2 adenosine receptors. The relative contributions of each receptor have not yet been established, but both receptor subtypes are in the brain and periphery (McLellan et al., 2016).

Adenosine inhibits the release of neurotransmitters in the brain. Therefore, adenosine receptor antagonists, like caffeine, promotes the release of neurotransmitters such as glutamate, serotonin, acetylcholine, noradrenaline and dopamine. Adenosine receptors, A1 and A2 have been associated with the behavioral effects of caffeine. For example, caffeine has been linked to aggression because its inhibitory effects on serotonin. It is also important to note that genetics as well as lifestyle can influence adenosine receptor number and sensitivity and can influence individual responses to a specific dose of caffeine (McLellan et al., 2016).

Regarding the central nervous system, caffeine enhances the release of neurotransmitters such as catecholamines, serotonin and acetylcholine associated with vasoconstriction in the brain and vasodilation in peripheral organs (Deslandes, Veiga, & Cagy, 2005). The pharmacological effects of caffeine consumption also include an increase in energy metabolism, decrease in smooth muscle contraction, vasodilation, positive inotropic effects on the heart, vasoconstriction, and increased blood pressure (Stohs & Vladimir, 2016). The increase in blood pressure may also elicit caffeine induced diuresis, vasodilation and sodium reabsorption in response to an increased 11 glomerular filtration rate (Stohs & Vladimir, 2016). It also decreases cerebral blood flow and increases brain metabolism (Deslandes et al., 2005).

Higher concentrations of caffeine increase intracellular cAMP which allows calcium release and increases cardiac contractility (Cappellitti et al., 2015). Caffeine also induces calcium release from the sarcoplasmic reticulum and inhibit its reuptake, causing increased contractility during submaximal contractions in habitual and nonhabitual caffeine consumers (Cappellitti et al., 2015). Intracellular calcium determines nitric oxide synthase activation; therefore, some effects of caffeine may be mediated by neuromuscular function and contractile force increases in skeletal muscle. Caffeine is also an inhibitor of phosphodiesterase. Cyclic adenosine monophosphate hydrolyzes phosphodiester linkages in molecules inhibiting their degradation.

Cyclic adenosine monophosphate stimulates lipolysis by activation of hormone sensitive lipase.

Hormone sensitive lipase is stimulated by phosphorylation mediated by protein kinase A. Protein kinase A phosphorylates numerous enzymes involved in glucose and lipid metabolism. The inhibition of phosphodiesterase requires very high doses of caffeine, that is unlikely present in a standard diet (Cappellitti et al., 2015).

Caffeine also alters substrate preference during exercise from glycogen to lipids by stimulating hormone sensitive lipase while inhibiting glycogen phosphorylase (Graham & Spriet,

1995). This increase in lipolysis may partly explain the improved performance sometimes seen during endurance exercise, and appears to be dose related (Graham & Spriet, 1995). Graham &

Spriet (1995) examined the effects of caffeine on exercise performance and metabolism in well- trained endurance athletes after they ingested various doses of caffeine. The study participants consumed either a placebo or caffeine (3, 6, or 9 mg/kg), 1 hour before treadmill running at 80%

VO2max until they reached volitional fatigue. Endurance performance was enhanced with both 3 and 6 mg/kg of caffeine, although there was no significant effect with 9 mg/kg of caffeine. At 12 rest caffeine elevated free fatty acids, but during exercise free fatty acid concentrations were elevated only in the 9 mg/kg group when compared to the control (Graham & Spriet, 1995).

Later, Daniels et al. (1998) evaluated resting and exercising study participants before and after the consumption of 6mg/kg of caffeine or a placebo. Both resting and exercise SBP and mean arterial pressure were significantly elevated. There were no significant findings regarding HR

(Daniels, Mole, Shaffrath, & Stebbins, 1998).

taurine.

Taurine is an amino acid found in high concentrations in the heart and skeletal muscle

(Schaffer, Jong, & Ramila, 2010). Monster Energy®, original flavor, 16oz can contains 2000 mg of taurine (Monster, 2013). There is no reason to believe caffeine and taurine would have any interaction while consuming an energy drink because they act on different receptors (Myers,

2003).

The majority of trials to date have looked at taurine in combinations with other ingredients, therefore it is difficult to determine the effects of taurine alone on healthy study participants. Data in the few clinical trials found are not conclusive enough to draw conclusions of taurine effects on energy metabolism. The effect of oral taurine supplementation (1,660 mg) was evaluated on endurance trained male cyclists 1 hour before exercise (Rutherford, Spriet, &

Stellingwerff, 2010). An increase in total fat oxidation (16%) was observed, but no effects were found on fluid intake, heart rate, exertion, or performance. In this study, the taurine supplementation was concluded to improve mitochondrial function, since mitochondria are the location of fatty acid degradation. Another study, Kammerer et al., examined taurine supplementation (1000 mg) on physical and mental performance in young soldiers (Kammerer,

Jaramillo, & García, 2014). No differences were found regarding the taurine group on cardiorespiratory fitness, concentration, and memory (Kammerer, Jaramillo, & Garcia, 2014). 13

Taurine has several physiological functions including: taurine depletion leading to the development of cardiomyopathy, osmoregulation, and modulation of protein kinases and phosphatases within the cardiomyocyte (Schaffer et al., 2010). Pion et al. (1987) was the first study to report the development of cardiomyopathy when cats were given a taurine deficit diet.

Taurine deficit cardiomyopathy is characterized by reduced fractional shortening combined with increased left ventricular chamber size, with an impaired response to dobutamine. The mechanisms of the development of taurine deficient cardiomyopathy have not been established.

However, it is important to note that heart failure is characterized by impaired contraction and the many conditions that cause heart failure are taurine dependent (diminished handling of calcium, impaired calcium sensitivity to proteins, loss of cardiomyocytes, and insufficient ATP). Taurine functions as an osmoregulator by normalizing cell volume in most cell types. Although mechanisms are unknown, the osmoregulatory activity of taurine does appear to be crucial to cell survival. In the heart, taurine-dependent phosphorylation of pyruvate dehydrogenase is promoted by reductions in taurine levels (Schaffer et al., 2010). Taurine depletion also leads to significant increases in lactate and pyruvate production (Mozaffari et al., 1986).

In a study aimed to investigate the influence of taurine on cardiac parameters before and after exercise, healthy study participants ingested the original “Red Bull” drink, a similar drink without taurine, or a placebo drink (Baum & Weib, 2001). Echocardiographic exams were conducted before drink consumptions, 40 minutes after, and post-exercise. Stroke volume was significantly increased in the Red Bull group (80.4+/-21.4 ml before drink vs. 97.5+/-26.2 ml) post-exercise (Baum & Weib, 2001). One limitation of this study is that glucuronolactone was included in the original “Red Bull” drink, but absent in the drink without taurine. Thus, this is an inherent weakness in their study as glucuronolactone and caffeine interaction may induce stroke volume changes. 14

l-carnitine.

L-carnitine is added in energy drinks to promote fat metabolism and increase endurance

(Seifert, 2010). Monster Energy® drinks contain an ‘energy blend’ (5000mg) containing: L- carnitine, glucose, caffeine, guarana, inositol, glucuronolactone and maltodextrin (Monster,

2013). L-carnitine is a cofactor of several enzymes including transferase necessary for the transformation of long chain fatty acids, and their transport into the mitochondrial matrix. B- oxidation of free fatty acids precedes their entry into the Krebs cycle. If there is an L-carnitine deficit, fatty acids will accumulate in the cytoplasm and produce a toxic effect as well as decreased energy (Goa, 1987). Ingestion of 2g L-carnitine and 80 g carbohydrate twice daily for

12 weeks by 14 male volunteers, who performed low and high intensity exercise 3 times a week, demonstrated increased work output by 11% and 55% less muscle glycogen utilization compared to controls (Wall, Stephens, & Constantin, 2011). The muscle phosphocreatine to ATP ratio was better maintained in the L-carnitine group when compared to the control group. The improvement in muscle phosphocreatine to ATP ratio is a result of muscle glycogen sparing, which is consistence with the increase in lipid utilization and an overall improvement in performance.

Regarding cardiovascular effects, L-carnitine supplementation has been associated with a significant reduction in all-cause mortality, ventricular arrhythmia, and angina in the setting of acute myocardial infarction (Shang, Sun, & Li, 2014). Metabolically, L-carnitine is a stereoisomer of dietary carnitine. Cardiac muscle cells cannot synthesize L-carnitine, and must acquire L-carnitine exogenously via a transporter. Cardiac mitochondria then import fatty acyl moieties for beta oxidation, the primary energy source in the heart muscle (Shang et al., 2014).

Therefore, L-carnitine deficiencies or its transporter have adverse effects on cardiomyocytes, resulting in cardiomyopathy, cardiac arrhythmia, cardiac insufficiency, and heart failure (Shang et 15 al., 2014). L-carnitine supplementation aids in resumption of normal oxidative metabolism and restoration of myocardial energy reserves. Thus, L-carnitine supplementation has therefore been shown to have favorable effects in cardiovascular patients.

BP Responses after ED Consumption

Caffeine consumption by individuals who do not regularly consume caffeine (caffeine naïve) causes an acute rise in BP, but tolerance develops rapidly and BP will return to baseline

(Myers, 2003). A few studies have found that ED consumption appears to acutely raise BP

(Grasser et al., 2014; Shah et al., 2016; Steinke et al., 2009). Steinke et al. (2009) observed changes in BP in 15 healthy study participants who consumed 500 mL of an energy drink. On day 1 of the study, baseline BP, HR, and ECGs were measured. Participants then consumed 500 mL (2 cans) of an energy drink and measurements were repeated 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours later. Participants then drank 500 mL of energy drink daily for the next 5 days. Day 1 protocol was repeated on Day 7 (Steinke et al., 2009). Within 2 hours SBP increased

10 mmHg and HR increased 5-7 beats per minute, although no significant ECG changes occurred

(Steinke et al., 2009). In a crossover study, Grasser et al. (2014) later examined 25 healthy study participants after consuming 355 mL of Red Bull® energy drink. The authors found that Red

Bull® consumption led to significant increases in SBP (114 ± 2 mmHg) and DBP (73 ± 1 mmHg)

20 minutes post drink consumption versus controls (Grasser et al, 2014). More recently, Shah et al. (2016) observed higher SBP in healthy volunteers who consumed 32 oz. (946 mL) of an energy drink versus a control drink. The authors found increased SBP only at 2 hours post ED consumption. (Shah et al., 2016).

Not all studies have seen ED-induced elevations in BP. For example, one study compared hemodynamic responses in healthy male study participants after they consumed Red

Bull®, Red Bull® (sugar free), and a caffeine control drink. Beat-to beat hemodynamic 16 measurements were made continuously for 30 minutes at baseline and for 2 hours following ingestion of 355 mL of the randomly assigned drink. There were no significant BP differences between the three groups (Miles-Chan et al., 2015). All three groups consumed caffeine so it is logical that there were no differences. Another study, Brothers, Christmas, Patik & Bahella

(2016) compared hemodynamic responses after study participants ingested either an energy drink, coffee, or water as a control, but found no effects on SBP or DBP between the three groups 6 hours post-consumption (Brothers, Christmas, Patik & Bahella, 2016). More recently, the effects of energy drink consumption on running economy and cardiovascular responses were assessed

(Peveler, Sanders, Marczinski, & Holmer, 2017). Fifteen recreational runners ingested either an energy drink or control drink before they completed 4 trials. The trials included one graded exercise test and 3 fifteen-minute economy trials. SBP was significantly higher in the energy drink group, when compared to the control drink (Peveler, Sanders, Marczinski, & Holmer,

2017). Consequently, BP seems to increase after ED consumption. More research is needed to thoroughly test BP responses after ED consumption employing a variety of conditions to include exercise, volume of energy drink consumed, duration of post-consumption measurements, and acute versus chronic ED ingestion. Also, caffeine dosing with study participants’ body weight could be normalized to mg of energy drink to kilogram of body weight.

HR Responses after ED Consumption

During exercise, resting HR should increase. As exercise workload increase, so does HR until maximal HR is reached at maximal workload. Heart rate increases due to a decrease in vagal tone and increase in sympathetic activation (Lim et al., 2016). During exercise, the increase in HR, along with increased stroke volume, increases the blood flow and oxygen supply to vital organs. To examine the effects of ED consumption on HR responses, Steinke et al.

(2009) had 15 healthy study participants consume 500 mL of an energy drink. Within 2 hours 17 there was a significant increase in HR (5-7 beats/min). In agreement with this study, Grasser et al. (2014) found significantly increased HR 2 hours after consuming 355 mL of an energy drink in 25 study participants. In contrast, Al-Fares et al. (2015) had 32 healthy females consume

4ml/kg of a standardized “Energy Drink” or control drink, 45 minutes prior to a graded maximal exercise test. Heart rate, BP, oxygen saturation, and blood lactate were recorded before and after the exercise. No significant differences were found between the placebo and energy drink groups

(Al-Fares, Alsunni, & Majeed, 2015). In the two resting studies, Grasser et al. (2014) and Al-

Fares et al. (2015), it can be concluded that energy drinks will cause an increase in heart rate 2 hours post ED consumption.

Basic Electrocardiography (ECG)

An ECG is a recording of the electrical activity of the heart. Each contraction of the heart is preceded by an excitation waves of electrical activity that originate in the sinoatrial (SA) node

(Khan, 2010). The SA nodes spontaneous depolarization and repolarization acts as an automatic pacemaker stimulus. These waves then spread through the atria and travel to the atrioventricular

(AV) node and to the bundle of His (Khan, 2010). The conduction then travels down the right and left bundle branches and to the Purkinje fibers.

A normal ECG has specific components that appear in waveforms. The first deflection from the zero baseline is the p wave, which usually has a positive deflection and smaller amplitude than other waves. This is because the p wave represents atrial depolarization. The atria are represented by a small wave amplitude when compared to the ventricles. The PR interval represents the time for the impulse from the atria to the AV node, bundle of His, bundle branches, and Purkinje fibers until the ventricles begin depolarization. The QRS complex is the next wave and is usually the largest in amplitude. The QRS complex represents ventricular depolarization. The ST segments lies between the end of the QRS complex and the beginning of 18 the T wave and represents the period where all parts of the ventricles are depolarizing. The T wave follows the QRS complex and represents ventricular repolarization. It is a broad rounded wave (Nishijima, Ikeda, & Takamatsu, 2002). The QT interval is measure from the beginning of the QRS complex to the end of the T wave.

Numerous different adverse cardiovascular events have been reported after the consumption of energy drinks (Goldfarb et al., 2014). However, studies like Fletcher et al., and

Alsunni et al. have highlighted the significant effect of energy drinks on the QT interval. This is critical because QTc prolongation more that 60 ms from baseline is a recognized marker of increased risk for fatal arrhythmias (Fletcher et al., 2017). For this reason, further energy drink studies evaluating the effect on QT interval should be conducted.

ECG Responses Following ED Consumption

While caffeine consumption alone appears to cause changes in HR and BP, it does not cause ECG changes (Steinke et al., 2009). However, some evidence suggests that ED consumption may result in ECG changes. For example, Alsunni, Majeed and Yar (2015) found a significant increase in the QTc interval in obese study participants (mean body mass index 34.5)

60 minutes after they consumed 5ml/kg body weight of an energy drink. These findings may be the result of the relative volume, with respect to body weight, consumed. The authors concluded further that the consumption of energy drinks by obese individuals could have a greater tendency toward autonomic imbalance, which could lead to dangerous effects on cardiac function. They suggested that this imbalance may be due to a reduction in vagal tone and an increase in sympathetic innervation (Alsunni et al., 2015). Fletcher et al. (2017) had participants consume

946 mL of an energy drink or a caffeine control drink to assess ECG and BP responses.

Measurements were obtained at baseline, 1, 2, 4, 6, and 24 hours post drink consumption. A significantly higher QTc interval was observed at 2 hours post drink consumption when 19 compared to the caffeine control group, but not at other time points (Fletcher et al., 2017). By contrast in a similar study, 500 mL of an energy drink was consumed and although QTc was increased by 2.4% 4 hours post energy drink consumption, this increase was not significant

(p=0.368) (Steinke et al., 2009). It can be concluded that there were no significant changed observed because in Steinke et al., participants consumed a much lower volume of energy drink.

Therefore, the discordant findings in these two studies could be due to the volume of energy drink consumption. Similar to Steinke et al. (2009), no ECG changes were observed after 5 hours when healthy study participants consumed single and multiple energy shots (Shah, 2016). The research evidence regarding ED consumption effects on ECG patterns is likely due volume of energy drink consumed. With high dosing of energy drinks there is generally an observed increase in QTc (> 5ml/kg). Consequently, due to differing methodologies the effects of ED consumption on the QT interval requires additional research.

QT Responses after ED Consumption

The QT interval represents ventricular depolarization and repolarization (Food and Drug

Administration, 2005). It is measured from the beginning of the QRS complex to the end of the T wave. A delay in cardiac repolarization is reflected in the ECG as QT interval prolongation. This delay favors the development of cardiac arrhythmias including torsade de pointes, as well as other ventricular tachyarrhythmias. A noted feature in torsade de pointes is the pronounced prolongation of QT interval prolongation the beat before the arrhythmia. Torsade de pointes can turn into ventricular fibrillation, which may lead to sudden death.

The QT interval has an inverse relationship with HR, and is often corrected to estimate the QT interval when HR = 60 b·min-1. There are currently five formulas that may be used for this correction: 1) Bazett (QTcB=QT/RR1/2); 2) Fidericia (QTcFri=QT/RR1/3); 3) Framingham

(QTcFra=QT+0.154 (1−RR)); 4) Hodges (QTcH=QT+0.00175 ([60/RR]−60)); and 5) Rautaharju 20

(QTcR=QT−0.185 (RR−1)+k (k=+0.006 seconds for men and +0 seconds for women) correction formula into a HR dependent value, referred to as the QTc interval (Vandenberk, Vandael, &

Robyns, 2016). Of these five choices, the Fridericia and Framingham correction formula showed the best rate correction and significantly improved the prediction of a 30-day and 1-year mortality in 6,609 adult patients during a 2-month period (Vandenberk et al., 2016). The authors found that the Bazett formula overestimated the number of patients with QTc prolongation.

A QT/QTc study can also be used to determine if a drug has an effect on cardiac repolarization. This would be detected on the ECG as QT/QTc prolongation. No significant increase in QT/QTc will allow researchers to evaluate a drug during stages of development. A significant increase in QT/QTc will elicit an ECG safety evaluation during the later stages of drug development (Guidance, 2005). QT/QTc >500ms is generally used a discontinuation parameter or threshold for concern. This is expressed as an absolute value while a change from baseline

>60ms is also used as a contraindication (Food and Drug Administration, 2005).

Long QT syndrome is an inherited disease caused by an abnormality in cardiac repolarization. This is reflected on the ECG as prolonged QT interval. The symptoms of Long

QT syndrome include prolonged QT interval, syncope, torsade de pointes, and sudden cardiac death. Syncope usually occurs in patients with prolonged QTc syndrome during exercise and without warning (Alders & Christiaans, 2003). There is biological research implementing that

Long QT syndrome is a result of mutations in the genes encoding for the sodium or potassium channel proteins. For example, KCNQ1 gene mutations have been determined to be responsible for over 50% of LQTS1 cases (Herber, 2002). Genetic testing can be done to detect a change in one of the 15 genes associated with QTc prolongation. The most common types of QTc are

LQT1, LQT2, and LQT3. These types of QTc prolongation are associated with the gene mutations (Alders & Christiaans, 2003). Initially, long QT syndrome prevalence was assumed to 21 be between 1/5000 and 1/20000, with no data to support these claims (Schwartz & Crotti, 2012).

The incidence of Long QT syndrome (Schwartz, Crotti, & Pedrazzini, 2009) is 1:2534

(0.00039%). This study included 44,596 healthy infants 15 to 25 days old. This value is higher than the incidence of an adverse cardiovascular event after the consumption of an energy drink

1:6,347,647 (0.000016%) (Goldfarb et al., 2014).

In the early 1990s, prolonged QTc was recognized as an independent risk factor for sudden death due to cardiac arrest (Graebe, 1991). More recently, a cohort study assessed the independent prognostic significance of the QT response compared to HR during post-exercise recovery (Johnson, Holly, & Goldberger, 2010). Patients were included in this study that were referred for treadmill exercise stress testing over a 5-year period. An abnormal QTc at 500 ms during recovery was found to predict all-cause mortality (Johnson et al., 2010). This article emphasized the importance of the QT interval as a prognostic marker during recovery because the

QT interval during recovery incorporates both repolarization and autonomic responsiveness

(Johnson et al., 2010). A meta-analysis of 23 studies assessed the relationship between QT interval and mortality endpoints and found interesting results (Zhang, Post, & Blasco-

Colmenares, 2011). The authors concluded that a strong association exists between prolonged

QT interval and increased risk of total, cardiovascular, coronary, and sudden cardiac death.

Therefore, the QT interval length appears to determine mortality in the general population

(Zhang, 2011). Another article, focusing on QTc behavior during exercise, highlights the importance of the identification of a prolonged QT interval (Schwartz & Crotti, 2010). Many patients that have LQTS may have a normal or a borderline prolonged QT interval during exercise, as an exercise-induced increase in HR may mask a prolonged QT interval. The authors suggested that QTc prolongation at 4 minutes of recovery after an exercise stress test could be used for optimal diagnosis of LQTS (Schwartz & Crotti, 2010). 22

Conclusion

There is limited evidence that ED consumption may alter cardiovascular responses and precipitate a cardiovascular event in asymptomatic individuals with ECG conduction abnormalities. Some of the cases of cardiovascular-associated adverse events were due to high consumption of EDs, or occurred in undiagnosed, high-risk individuals. Some evidence demonstrates that ED (>5ml/kg) consumption may prolong the QT interval, but this needs to be further studied. Individuals with Long QT syndrome may be at an elevated risk for arrhythmias after ED consumption. Energy drink-induced prolongation of the QT interval during recovery from exercise may be the cause of sudden death.

23

Chapter 3

Methods

Study Design

The purpose of this study was to determine if the consumption of a 24oz can of Monster

Energy® drink (709 mL) prior to exercise would affect QTc during a maximal graded exercise test and 2-hours post exercise. This study employed a double-blinded, randomized, crossover design to evaluate the cardiovascular effects of Monster Energy® drink. A secondary purpose was to compare ED versus control drink on maximal exercise performance.

Study Participants

All study participants were briefed about the details of the study, presented with the

Informed Consent Document (ICD) and HIPAA documents and asked to sign the appropriate forms voluntarily if they wished to be a part of this study. The study participants included in the study were healthy male and female CSUS students. They were between 18 to 40 years old. The study participants refrained from caffeine and energy drink use 48 hours prior to study days 1 and

8.

In an effort to recruit study participants in general good health, those with the following medical conditions or disease states were excluded from enrollment: cardiovascular risk factors including a heart rhythm other than normal sinus, history of atrial or ventricular arrhythmia, family history of premature sudden cardiac death before the age of 60, left ventricular hypertrophy, atherosclerosis, hypertension, palpitations, T-wave abnormalities, baseline corrected

QT (QTc) interval greater than 440 milliseconds(ms) (this was determined on the ECG obtained during initial screening appointment), blood pressure at initial screening appointment or at baseline on study Day 1 greater than 130/80, the presence of any known medical condition confirmed through participant interview, concurrent use of ANY medication taken on a daily 24 basis (daily basis is defined as greater than 2 days per week), pregnant or lactating females

(pregnancy test performed before each experimental session, days 1 and 8), all non-English speaking / writing study participants were excluded from the study due to unavailability of medical qualified translator. If the study participant refused to sign the informed consent document or HIPAA Authorization they were also excluded from the study. Study participants who met inclusion criteria (a normal sinus rhythm and BP < 130/80 mmHg), and were approved by the cardiologist were enrolled in the study and allocated a randomized participant ID number.

They were also given the “Participant Instruction Sheet” that outlined the study’s procedures.

Procedures

All study participants were briefed about the details of the study, presented with the

Informed Consent Document (ICD) and HIPAA documents and asked to sign the appropriate forms voluntarily if they wished to be a part of this study. Upon obtaining consent, study participants were further screened to see if they qualified for inclusion in the study. The questions included an inquiry into the usual caffeine intake of the participant, to assess whether they were caffeine naïve or caffeine tolerant. The participant’s height, weight, and ethnicity were recorded. A resting 12-lead baseline ECG and BP were obtained to rule out cardiovascular abnormalities. A cardiologist reviewed every baseline ECG and BP reading, and study participants were only included in this study if they had a normal sinus rhythm and a BP < 130/80 mmHg.

After study enrollment, participants were randomly assigned to consume either a single

24oz container of a Monster Energy® drink, or a 24 oz container of a control drink. Each control drink was comprised of 585mL of carbonated water, 30 mL of reconstituted lime juice, and

105mL of cherry flavoring. This control drink formula has been used previously (Fletcher et al. 25

2017). The carbohydrate content of the 24oz control drink was 81g, which is the same as the 81g per 24oz Monster Energy® drink.

Blinding and randomization was performed by a member of the David Grant Medical

Center, Travis AFB, CA pharmacy staff who was not in contact with participants during the experiments and retained the randomization code in a sealed envelope in his office. The PI, AIs, and SC were blinded. The Monster Energy® and control drinks were repackaged in the David

Grant Medical Center pharmacy in identical 24oz opaque bottles. All drinks were made within 7 days prior to being consumed and stored at 3-4°C until consumption. Study drinks were readily available for an investigator/coordinator for distribution to the participants.

Throughout the study, study participants visited the Sac State exercise physiology laboratory. The study participants visited twice separated by 4-14 days to undergo testing in the experimental and control drink conditions. Prior to reporting to the lab, study participants had abstained from caffeine consumption for the previous 48-hour period. Study participants were also asked to abstain from vigorous strength and endurance exercise for the previous 24 hours prior to reporting. During each visit, ECG electrodes were placed on the upper body torso and remained in place throughout the experiment. For blood sampling purposes, a Teflon intravenous catheter was inserted into a forearm vein under sterile conditions and remained in place throughout the duration of the experiment. Baseline 12-lead ECG, HR, BP, blood sample, and body weight were obtained prior to consumption of assigned study drink.

Immediately following baseline measurements, study participants were given a 24oz bottle of Monster Energy® or control drink and asked to consume it within 20 min. After drink consumption and at the end of the 20 min period, the study participants performed a continuous, progressive bicycle ergometer test until volitional exhaustion. Study participants began cycling on a stationary cycle ergometer (Lode Excalibur, Lode, Groningen, Netherlands) at a resistance of 26

100 W for two minutes with the resistance increased by 25 W and 15 W for males and females, respectively, each minute thereafter. The study participants were encouraged to perform their best during testing. Since oxygen consumption was not measured during testing, maximal effort was considered achieved when the following two criteria were met: 1) attainment of 85% age- predicted maximal HR; and 2) volitional exhaustion. Estimated maximal HR was calculated using the Tanaka formula (Tanaka et al., 2001). The 12-lead ECG chest electrode remained in place on each study participants and was continuously monitored during exercise. During exercise, Rating of Perceived Exertion (RPE) was recorded at every minute just prior to each workload increase.

Table 1

Summary of Procedures During each Experiment

Measurement Baseline Drink Exercise Exercise 1, 4, 7 30 min 60 min 90 min 120 termination & 10 post- post- post- min min exercise exercise exercise post- post- exercise exercise ECG X X X X X X X HR X 20 15-20 X X X X X BP X mins mins X X X X X Body weight X

Immediately following completion of exercise, the study participants remained seated on the ergometer while ECG, BP, HR were recorded and blood samples were taken. The study participants remained seated on the ergometer approximately 10 minutes to observe and record

ECG at 1, 4, 7 and 10 min after exercise. After the 10 minute ECG monitoring period, the study participants remained in the lab, but were free to sit, stand or move around quietly. During the 2- hour post-exercise monitoring period, the study participants ECG, HR, and BP, were recorded at

30 minute intervals. Five minutes prior to each recording, the study participants were asked to sit quietly. In addition to blood sample collection immediately upon cessation of exercise, blood 27 samples were taken again at one and two hours post-exercise. Blood samples were measured for caffeine, taurine, L-carnitine, plasma free fatty acids, glucose, and lactate.

Data Analysis

The FDA advises that studies determining the presence of QT interval prolongation conduct a crossover or parallel group study and for it be powered enough to detect a 10msec change (Food and Drug Administration, 2005). A standard deviation of 14msec has been observed in previous work (Shaw, 2016).

Based on a mean change in QT/QTc interval of 10msec and a standard deviation of 14

(p=0.05 and 80% power) a sample size of 18 study participants were required for this study. Due to screening failures and study participant dropout, 8 study participants completed the study. A two-factor repeated measures ANOVA was used to ascertain TIMEPOINT (Baseline, Max, 30,

60, 90, and 120 minutes post exercise) and DRINKTYPE (Energy Drink vs. Placebo) interactions for QT/QTc interval, Systolic BP, Diastolic BP, HR, and blood metabolites. It must be noted that the assumptions for performing a repeated measures ANOVA were not met, therefore results should be interpreted with caution (although the Greenhouse-Geisser correction was used to adjust the p-value for the violation of the assumption of sphericity). A paired t test was performed to determine if there was a difference in the maximum exercise time and maximal power (measured in Watts) at VO2 Max between the ED and placebo groups. A paired t test was performed to determine if there was a difference in estimated relative VO2 Max values between the ED and the placebo groups. Estimated VO2 Max values were calculated using the metabolic equation for cycling from ACSM’s Guidelines for Testing and Exercise Prescription (Pescatello,

2014). The significance level was set at p = 0.05 for all analyses and Bonferoni adjustments were made for all paired t-tests. All data is presented as mean ±SD. STATA/SE 14.2 for Windows

(College Station, TX) was used for the analyses. 28

Chapter 4

Results

Study Participant Characteristics

Three men and 5 women (n=8) participated in the study. Seven of the study participants were Caucasian and one study participant was Asian. Seven of the study participants were caffeine tolerant (caffeine consumption daily) and 1 study participant was caffeine naive (≤ caffeine consumption 2 times per week). Mean age, weight, and height were 24.3±5.8 years,

169.7±9.3 cm, and 63.1±9.4 kg.

Table 2

Summary of Study Participant Characteristics (n=8, values displayed as mean ± SD)

Age 24.3±5.8 (years) Height 169.7±9.3 (cm) Weight 63.1±9.4 (kg)

29

Systolic Blood Pressure

Energy Drink consumption had no significant effect on SBP at any time interval compared to the placebo. As expected, SBP at maximal exercise was found to be significantly higher

(p<.0001) compared with any other time point (Fig. 2).

170 * Energy 160 drink 150 Placebo 140 130

SBP(mm Hg) 120 110 100 Baseline Max 30 min 60 min 90 min 120 min TIME

Figure 2. Pre-, max, and post-exercise systolic blood pressure following drink consumption.

*denotes ED and placebo different from baseline, 30, 60, 90 and 120 min (p<0.05).

30

Diastolic Blood Pressure

Energy Drink consumption had no significant effect on DBP at any time interval compared to the placebo. As expected, DBP at maximal exercise was found to be significantly higher

(p<.0001) compared with any other time (Fig. 3).

85 Energy * drink 80 Placebo * 75

70 DBP(mm Hg)

65

60 Baseline Max 30 min 60 min 90 min 120 min Time

Figure 3. Pre-, max, and post-exercise diastolic blood pressure following drink consumption.

*denotes placebo different from baseline, 30, 60, 90 and 120 min (p<0.05).

31

Heart Rate

No statistically significant effect between ED consumption vs. placebo was found on heart rate. However, HR during exercise and post-exercise recovery significantly different vs. baseline HR within each group (p<0.05) (See Fig. 4).

200 Energy 180 ** drink Placebo 160 *** 140 **** ***… 120 ****… 100 Heart (BPM) rate * 79 80

60 Baseline Max 1 min 4 min 7 min 10 min 30 min 60 min 90 min 120 min Time

Figure 4. Pre-, max, and post-exercise heart rate following drink consumption.

*denotes ED and placebo different from Max, 1 min, 4 min, 7 min, 10 min, 30 min, 60 min, 90 min, and 120 min (p<0.05). **denotes ED and placebo different from Baseline, 1 min, 4 min, 7 min, 10 min, 30 min, 60 min, 90 min, and 120 min (p<0.05). ***denotes ED and placebo different from 4 min, 7 min, 10 min, 30 min, 60 min, 90 min, and 120 min (p<0.05). ****denotes

ED and placebo different from 7 min, 10 min, 30 min, 60 min, 90 min, and 120 min (p<0.05).

*****denotes ED and placebo different from 10 min, 30 min, 60 min, 90 min, and 120 min

(p<0.05). ******denotes ED and placebo different from 30 min, 60 min, 90 min, and 120 min

(p<0.05).

32

Rate of Perceived Exertion

Since study participants complete their exercise tests at different stages, only stages completed by all eight study participants (1-4) were statistically analyzed for Rate of Received

Exertion (RPE). No effects were found on between the ED and the placebo groups on RPE, as well as no interaction of the ED and the placebo groups and time.

Table 3

RPE Values presented as mean ± SD

Stage 1 2 3 4

Energy Drink 8.1±1.9 9.8±2.3 11.4±2.7 12.4±2.8

Placebo 8.5±2.1 10.1±2.7 11.5±2.9 13.0±3.3

33

Exercise Time

There was significantly increased (p=.02) time to maximal exercise exhaustion when receiving the ED compared to the placebo (0.4 minutes). A significant difference was observed in time to maximal exercise exhaustion in the ED group vs. the placebo group (p<0.05). Study participants rode a mean 0.4 minutes longer in the ED group vs. the placebo group.

9.80

9.60 *

9.40

9.20

9.00 Energy Drink Placebo 8.80

Exercise Exercise (minutes) time 8.60

8.40

8.20 Energy Drink Placebo

Figure 5. Time to exhaustion at maximal exercise between trials. *denotes significantly higher vs. placebo (p<0.05).

34

Maximal Power

A significantly higher maximal power was achieved in the ED group vs. the placebo group (p<0.05). The ED group achieved a mean max power output of 243.75 ± 92.22 W vs.

233.75 ± 90.62 W in the placebo group.

255

250 *

245

240

235 Energy Drink

Placebo

230 Maximal Maximal Power (W) 225

220

215 Energy Drink Placebo

Figure 6. Maximal Power (W) in the ED and Placebo groups. *denotes significantly higher vs. placebo (p<0.05).

35

Relative VO2 Max

No significant difference was found in relative VO2max between the ED and the placebo groups (p=0.05).

51

50

49

48

47 Energy Drink Placebo 46

45 Estimated (ml/kg/min) VO2 44

43 Energy Drink Placebo

Figure 7. Relative VO2 (ml/kg/min) in the ED and Placebo groups. Estimated VO2max values were calculated from the equation VO2(ml/kg/min) =3.5+3.5+((1.8*Power)/Mass).

36

QT/QTc Interval

No significant difference was found between the ED and the placebo group at rest and at max exercise (p=0.05). However, QTc in placebo and ED group during max exercise was significantly different compared to any other time within each group (p<0.05) (See Fig. 8).

390

370

350 * msec 330 Energy drink 310 * Placebo 290 Baseline Max 1 min 4 min 7 min 10 min 30 min 60 min 90 min 120 min Time

Figure 8. Pre-, max, and post-exercise QTc (msec) following drink consumption.

*denotes placebo different from baseline, 1 min, 4 min, 7 min, 10 min, 30 min, 60 min, 90 min, and 120 min (p<0.05).

37

Chapter 5

Discussion

The purpose of this study was to examine exercise and post-exercise QTc responses after consuming an energy drink (709 mL). The major finding of this study was that the QTc at maximal exercise decreased across both groups. However, there was a non-significant trend where the ED group decreased by approximately 40 msec versus the placebo group which had a

70 msec drop compared to rest. In addition, ED consumption resulted in longer exercise time

(0.4 min) and higher maximal power output (243.75 ± 92.22 W) compared to the placebo group

(233.75 ± 90.62 W). Energy drink consumption appeared to result in no other significant physiological differences during maximal exercise and 2 hours of recovery.

We did not find a significant difference in QTc when study participants consumed a dosage of energy drink of 709 mL compared with the placebo group. A few resting studies have found ED consumption to adversely impact the QTc interval (Fletcher et al., 2017 and Alsunni et al., 2015). In Steinke et al., 2009, participants consumed 500 mL of an energy drink and found a

2.4% non-significant increase in QTc (9.8 msec) during rest which is more than the changes in

QTc in our study. In Alsunni et al. (2015), a significant 4.7 % increase (16.6 msec) in QTc interval 60 minutes post consumption was found during rest, but in obese study participants. In

Alsunni et al. (2015), the study participants consumed 5ml/kg of energy drink relative to body weight. In Fletcher et al. (2017), participants consumed 946 mL of an energy drink, and found a significantly higher QTc interval (10 msec) 2 hours post consumption. The dosage in the current study (709 mL) was enough to cause a significant increase in performance but did not alter other cardiovascular physiological parameters significantly (SBP, DBP, HR and QTc) during maximal exercise nor 2 hours post-recovery. 38

QT/QTc interval prolongation favors the development of cardiac arrhythmias and is recognized as an independent risk factor for sudden death due to cardiac arrest (Graebe, 1991). In our study, the QTc interval was statistically lower at maximum exercise in both groups compared to rest and recovery. However, there was a nonsignificant trend in that the ED group which had a smaller decrease in QTc during max exercise than the placebo group. Although this nonsignificant difference was small, this finding may be meaningful for some groups sensitive to

QT interval disturbances, such as those with undiagnosed Long QT Syndrome Type 1 (LQT1).

Indeed, individuals with LQT1 already exhibit paradoxically longer QT intervals at high-intensity exercise that presents a higher risk of a cardiac event (Koonlawee, 2012). Therefore, based on our observation, ED consumption could potentially exacerbate QT prolongation during high- intensity exercise in LQT1 individuals. With forty-two percent of adolescents reporting consuming an energy drink in a two-week period (Odea, 2003), and 17 adverse events reported after energy drink consumption (Goldfarb et al., 2014), our study may indicate a possible link between ED consumption and QT prolongation during exercise in LQT1 study participants.

However, the negative physiological risk associated with ED use during maximum exercise and recovery is not supported by the evidence in this study since the QTc decreased in comparison to rest and recovery and was well below threshold to induce arrhythmias (increase in resting QTc >

60 msec) and no adverse effects were observed for HR and BP.

Energy drink consumption resulted in a significantly (4.4%) improved (0.4 min) time to exhaustion during the maximal exercise test. Caffeine is a known ergogenic aide that improves endurance performance. For instance, in Graham and Spriet (1995), (treadmill running at 80%

VO2max until volitional fatigue) exercise time increased by 22% (49.4 minutes) after caffeine consumption of 3 mg/kg, 6 mg/kg but not 9 mg/kg. They found elevated free fatty acids at rest, and during exercise in the 9 mg/kg group when compared to the control. Caffeine alters substrate 39 preference during exercise by increasing lipid utilization by stimulating hormone sensitive lipase.

The increase in lipolysis could explain the improved endurance performance after caffeine consumption. Ivy et al. (2009) observed a ~3 min longer ride time to exhaustion (75% Wmax at

90 rpm) in 12 trained cyclists who consumed 500 mL of Red Bull energy drink (2.0 g taurine,

1.2 g glucuronolactone, 160 mg caffeine) vs. a placebo drink. Although the mechanisms of action of caffeine are not completely understood, the most convincing mechanism is the antagonistic effect of caffeine on A1 and A2 adenosine receptors in the central nervous system.

Caffeine has the ability to increase neural activity in the spinal cord, improving overall motor performance (De Morree, Klein, & Marcora, 2014). In De Morree et al. (2014), acute caffeine consumption decreased the primary motor activation required during knee extensions.

Study participants had a significantly higher maximum power (10 Watts) after consuming the energy drink, when compared to the placebo. The effects of caffeine (244 mg) on skeletal muscle may also be a contributing factor to significantly increasing VO2max power output.

Kalmar & Cafarelli (1999) examined the effects of caffeine on neuromuscular function in 12 male volunteers who performed 6 submaximal contractions at 50% of maximal voluntary contraction to fatigue after caffeine consumption compared to a placebo. The authors found that voluntary activation at maximal voluntary contraction was significantly higher (p<0.01) compared to the placebo. In addition, time to fatigue was increased after caffeine consumption (p<0.05) compared to the placebo. Caffeine may block the inhibitory effects of adenosine, which could increase the excitability of a motor unit (McLellan et al., 2016). Input to the alpha motor neuron may increase and then be closer to threshold and maximal activation (McLellan et al., 2016). Increasing activation may be apparent through increases in discharge rates and an increase in motor unit recruitment (Kalmar & Cafarelli,1999). Our findings (4.4% increase in time to exhaustion), in agreement with Kalmar & Cafarelli (10.8% increase in time to exhaustion) demonstrate that time 40 to exhaustion is increased after caffeine. However, our study participants did not have a significantly higher estimated VO2 max when comparing the energy drink group with the placebo. Failure to see this significant increase in estimated VO2max could be because we did not discriminate between active and sedentary individuals for this study. VO2max power was determined based on the watts performed before the study participant ended the test. This is a limitation because time was not considered in the VO2max estimation. Sensitivity of the measurement could have been lost due to a study participant stopping the test at the beginning versus the end of the stage. One limitation to using cycling as the exercise modality was that study participants were not trained and failed to reach maximum volitional fatigue. According to

ACSM Guidelines (Pescatello, 2014) the estimated VO2max values recorded from our study seem to be low, and this could be due to not achieving VO2max because of peripheral muscle fatigue.

In contrast to previous studies, we did not observe significant changes in SBP (Steinke et al., 2009, Shah et al., 2016 and Peveler et al., 2017). Steinke et al. (2009) observed a significant increase in SBP (7.9% on day one and 9.6% on day 7 of the study) four hours post consumption and DBP increased (7.0% on day one and 7.8% on day 7) 2 hours post energy drink consumption at a lower dose (500 mL). Also, Shah et al. (2016), found a significant increase in SBP (6 mmHg) 3 and 5 hours post consumption and DBP (4 mmHg) 1 and 5 hours post consumption of an energy drink at a higher dose (946 mL). In Peveler et al. (2017), study participants completed

3, 15-minute economy trials. During the economy trials SBP was found significantly higher in the energy drink group by 4.95% (Red Bull average increase = 8.18 ± 6.83 mmHg, Monster average increase = 9.54 ± 6.79mmHg, and 5hr-drink average increase = 8.07 ± 8.87 mmHg) after ingestion of one of three energy drinks (248.42 mL Red Bull, 473.18 mL Monster, and 27.50 mL

5 Hour Energy). No significant differences were found in DBP between the placebo, Reb Bull,

Monster and 5hr-drink. It was interesting that in this study the participants ingested a higher 41 volume of ED but there was no significant difference in the energy drink group and placebo SBP responses compared to Peveler et al. (2017). When comparing our results with the resting studies (Steinke and Shah), we did not record SBP and DPB up to 5 hours post-exercise. On the contrary, Peveler et al., found significant SBP after the 15-minute economy trials.

Energy drink consumption did not significantly affect HR during or after exercise. It is logical that the HR at maximal exercise was significantly higher than each other time point because as exercise workload increases, so does HR until maximal HR is reached at maximal exercise. In Steinke et al. (2009) and Grasser et al. (2014) HR was found significantly increased by 7.8% and 7 beats/minute two hours after consuming an energy drink (500 mL and 355 mL).

The ED volume consumed in this study (709 mL) was higher than these two studies. In agreement with our study, Al-Fares et al. found no significant differences in HR between the placebo and the consumption of 4ml/kg of an energy drink during and after exercise. In the current study subjects consumed a much higher dose (between 8.55 ml/kg to 13.14 ml/kg) and still found no significant effect on HR during or after exercise. The increase in sympathetic activation and decrease in vagal tone due to exercise may be greater than the increase in sympathetic activation after the consumption of an ED up to 13.14 ml/kg.

There were several limitations to this study. First, the sample size (n=8) was likely too small to detect changes in any of the parameters we measured. The a-priori statistical power was calculated using QT/QTc interval of 10msec and a standard deviation of 14 resulting in 18 study participants required to achieve meaningful statistical power (p=0.05 and 80% power). The current power of the study with 8 study participants was 46%. Another limitation in this study was that both caffeine naive and caffeine tolerant study participants were included in this study.

One study participant in this study was considered caffeine naive, while 7 study participants were considered caffeine tolerant. Although we attempted to normalize caffeine tolerance in all study 42 participants by requiring no caffeine consumption 48 hours prior to each experiment, it is possible this was insufficient time. It is also important to note that some of the study participants had difficulties consuming 709mL of the energy drink, and felt sick prior to exercise. This could have had a negative effect on their exercise test in the ED group. It may have been better to normalize the energy drink volume to body weight to ensure appropriate dosing. There was a wide range of

ED doses ingested by the study’s participants, 8.55 ml/kg to 13.14 ml/kg, since all participants ingested one 709 mL bottle of ED.

For future research, Holter monitoring should be employed to measure changes in QTC and rate of other arrhythmias at rest and after exercise with ED consumption. Also, the power of this study was limited and this could be improved with a larger sample size and longer measurement period (48 hours) with ED consumption before and after exercise. In addition, most of the available literature focuses on ED consumption in resting healthy study participants; therefore, more research is warranted on the combined effects of exercise and ED consumption in high risk populations such as LQT1 patients. Future studies should also evaluate potential gender and ethnicity differences after ED consumption. While the acute effects on QTc after energy drink consumption are important to investigate, the chronic consumption of energy drinks are likely equally as important to be investigated. It is possible the development of arrhythmias may be caused by chronic consumption of energy drinks opposed to acute consumption (Peveler,

Sanders, Marczinski, & Holmer, 2017).

Conclusion

Our findings indicate that 709 mL of ED consumption significantly improves time to exhaustion of a VO2max test by 0.4 minutes (4.4%). However, there was no significant cardiovascular differences in the QTc interval, SBP, DBP, and HR following ED consumption at rest, during maximal exercise and 2 hours post-exercise in this this study compared to the placebo 43 in this study. However, at max exercise there was a non-significant trend where the QTc decreased less than the placebo group. EDs should still be consumed with caution in order to prevent an increased QT interval in at-risk population such as those with undiagnosed Long QT

Syndrome Type 1 (LQT1). Although ED consumption within 2 hours did not significantly affect

SBP, DBP, HR and QTc during and after a max exercise test in healthy adults in our study, further research might explore a larger group of study participants, different volumes of EDs, longer measurement time of parameters before and after the ED is consumed, and type of exercise and type of recovery after ED consumption on the incidence and development of arrhythmias.

Based on this study, ED consumption is safe in healthy individuals 18-40 years old during and after exercise. A QTc change from a resting baseline >60ms is expressed as a threshold for concern, and the QTc change from baseline in our study was within normal limits and decreased by 44.8 ± 22.1 msec during maximum exercise.

44

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