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Nutrients 2020, 12, 654; doi:10.3390/nu12030654 www.mdpi.com/journal/nutrients
Article
Safety of Short-Term Supplementation with
Methylliberine (Dynamine
®
) Alone and in
Combination with TeaCrine
®
in Young Adults
Trisha A. VanDusseldorp
1,
*, Matthew T. Stratton
2
, Alyssa R. Bailly
1
, Alyssa J. Holmes
1
,
Michaela G. Alesi
1
, Yuri Feito
1
, Gerald T. Mangine
1
, Garrett M. Hester
1
, Tiffany A. Esmat
1
,
Megan Barcala
1
, Karleena R. Tuggle
3
, Michael Snyder
1
and Andrew S. Modjeski
1
1
Department of Exercise Science and Sport Management, Kennesaw State University,
Kennesaw, GA 30144, USA; tvanduss@kennesaw.edu (T.A.V.); abailly@students.kennesaw.edu (A.R.B.);
alyssajh@hotmail.com (A.J.H.); malesi@students.kennesaw.edu (M.G.A.); yfeito@kennesaw.edu (Y.F.);
gmangine@kennesaw.edu (G.T.M.); ghester4@kennesaw.edu (G.M.H.); tesmat@kennesaw.edu (T.A.E.);
megan.barcala@choa.org (M.B.); snyderme@mac.com (M.S.); modjeskia@gmail.com (A.S.M.)
2
Kinesiology and Sport Management, Texas Tech University, Lubbock, TX 79409, USA;
matthew.stratton@ttu.edu
3
Southern Regional Physician Management Group, LLC, Riverdale, GA 30274, USA; aneelrak@hotmail.com
* Correspondence: tvanduss@kennesaw.edu; Tel.: 1-470-578-4266
Received: 6 February 2020; Accepted: 25 February 2020; Published: 28 February 2020
Abstract: Methylliberine (Dynamine
®
; DYM) and theacrine (Teacrine
®
; TCR) are purine alkaloids
purported to have similar neuro-energetic effects as caffeine. There are no published human safety
data on DYM, and research on TCR is limited. The purpose of this study was to examine the effect
of four weeks of DYM supplementation with and without TCR on cardiovascular function and
blood biomarkers. One-hundred twenty-five men and women (mean age 23.0 yrs, height 169.7 cm,
body mass 72.1 kg; n = 25/group) were randomly assigned to one of five groups: low-dose DYM (100
mg), high-dose DYM (150 mg), low-dose DYM with TCR (100 mg + 50 mg), high-dose DYM with
TCR (150 mg + 25 mg) , and placebo. Regardless of group and sex, significant main effects for time
were noted for heart rate, systolic blood pressure, and QTc (p < 0.001), high-density lipoproteins (p
= 0.002), mean corpuscular hemoglobin (p = 0.018), basophils (p = 0.006), absolute eosinophils (p =
0.010), creatinine (p = 0.004), estimated glomerular filtration rate (p = 0.037), chloride (p = 0.030),
carbon dioxide (p = 0.023), bilirubin (p = 0.027), and alanine aminotransferase (p = 0.043), among
others. While small changes were found in some cardiovascular and blood biomarkers, no clinically
significant changes occurred. This suggests that DYM alone or in combination with TCR consumed
at the dosages used in this study does not appear to negatively affect markers of health over four
weeks of continuous use.
Keywords: TeaCrine
®
, Dynamine
®
, theacrine; methylliberine; purine alkaloid
1. Introduction
Caffeine is the most widely consumed alkaloid in the world. Its acute, positive impact on
concentration, mood, alertness, fatigue, pain/soreness perception, and exercise performance was well
studied (for reviews, please see References [1,2]). Chronic, regular caffeine consumption may blunt
the subsequent physiological effects of supplementation, likely through an increase in adenosine
receptor concentration [3,4], thereby reducing caffeine’s stimulatory effects [5]. Furthermore, high
dosages of caffeine may cause anxiousness and adverse neuroendocrine and cardiovascular effects
[6]. As such, there is a great deal of interest in caffeine alternatives that may promote similar ergogenic
outcomes, without these undesirable side effects.
Nutrients 2020, 12, 654 2 of 20
In addition to caffeine (1,3,7-trimethylxanthine), the purine alkaloids theacrine (1,3,7,9-
tetramethyluric acid) and methylliberine (O(2),1,7,9-tetramethylurate) were identified in the seeds
and leaves of various Coffea species [7,8]. This includes Coffea arabic and Coffea canephora. Theacrine
was also been found in Coffea liberica, Coffea dewevrei, Coffea abeokuta [8–10], and kucha tea (C. assamica
var. kucha (green tea)). Initially, caffeine accumulates in young leaves but it is gradually replaced by
theacrine and liberine. Due to purported synthesis from caffeine in some plants and structural
similarities to caffeine [5,11], both theacrine and methylliberine are theorized to produce similar
physiological effects with less side effects, potentially due to their different affinities with adenosine
receptors [11,12]. Although theacrine was first discovered in 1937 [13], little research (to our
knowledge, only seven studies) was conducted on theacrine’s effects on human health and
performance until recently [14–20]. Previous cell and animal model investigations on theacrine
reported improved antioxidant capacity [20] and anti-inflammatory responses [21], as well as
analgesic [21], antidepressive [22], and sedative/hypnotic [23] mood states. Feduccia and colleagues
also demonstrated that theacrine serves as an adenosine receptor antagonist and enhances
locomotion, likely attributed to theacrine’s effects not only on adenosine receptors, but also the
dopaminergic systems [24]. Interestingly, despite theacrines’s similarities with caffeine, Feduccia et
al. (2012) found that seven days of 48 mg/kg theacrine intraperitoneal injections did not induce
sensitization or negatively affect tolerance [24], which is typical with chronic caffeine consumption.
In contrast, less in known about methylliberine, which is hypothesized to have similar physiological
properties as caffeine and theacrine. To date, the only study on methylliberine examined the effects
of a >98.0% pure supplement (Dynamine®, Compound Solutions, Inc., Carlsbad, CA; referred to as
DYM), in Wistar rats [25]. Following a 90-day supplementation period, dose–response toxicology
evaluation reported no toxicologically relevant clinical effects or effects on clinical hematological
parameters [25]. Thus, both theacrine and methylliberine appear to be safe in animal models, but
information regarding their effect in humans is limited.
In 2016, a human study was published on TeaCrine® (Compound Solutions, Inc., Carlsbad, CA;
referred to as TCR), a commercially available, chemical equivalent bioactive version of theacrine. The
report indicated that eight weeks of supplementing with either 200-mg or 300-mg doses of TCR did
not negatively affect blood or hemodynamic measures associated with clinical safety, and positively
affected cholesterol [17]. The authors suggest that TCR may be a viable “nutraceutical” alternative to
cholesterol-lowering drugs, and, of late, a great deal of research on “nutraceuticals” and “functional
foods” (e.g., zinc, quercetin, resveratrol, grape-seed extract, black/green tea) that may reduce the
burden of dyslipidemia and cardiovascular disease exists (for reviews, please see References [26,27]).
However, no human research was conducted to date on methylliberine. Therefore, we sought to
investigate the safety of orally consumed, commercially available methylliberine in humans, as well
as expand on the limited information on theacrine by examining its combination with methylliberine.
Specifically, the purpose of this study was to examine the effect of four weeks of DYM
supplementation with and without TCR on parameters of cardiovascular function and blood
biomarkers associated with health. As methylliberine was never studied in humans, a four-week time
period was selected based on previous studies on novel dietary supplements [28–30]. Based on
previous findings [14,17,25], we hypothesized that DYM alone or with TCR would not produce
significant abnormal changes in cardiovascular function parameters of heart rate, blood pressure, or
electrical conduction of the heart assessed via an electrocardiogram, nor hematological markers of
health for both men and women.
2. Materials and Methods
2.1. Experimental Design
Apparently healthy males and females took part in this placebo-controlled, double-blind
investigation. Participants visited the laboratory fasted on three occasions: pre-participation
screening (Visit 1), Visit 2, and Visit 3. Visit 2 and Visit 3 were separated by four weeks of once-daily
supplementation. Participants were randomly assigned to one of five groups: low-dose DYM (100
Nutrients 2020, 12, 654 3 of 20
mg), high-dose DYM (150 mg), low-dose DYM with TCR (100 mg + 50 mg), high-dose DYM with TCR
(150 mg + 25 mg), and 125 mg of maltodextrin (placebo). Pre- and post-supplementation assessments
included measures of cardiovascular function (i.e., resting heart rate, resting blood pressure, resting
electrocardiogram (ECG)) and blood collection for the analysis of complete blood count (CBC),
complete metabolic panel (CMP), and lipid panel. Prior to all visits to the laboratory, participants
were asked to arrive in a fasted state (≥8 h), as well as abstain from caffeine (≥24 h) and exercise (≥24
h). The study was approved by the Kennesaw State University (KSU) Institutional Review Board (IRB
#18-223). All data were collected in accordance with the Declaration of Helsinki. An overview of the
study design may be seen in Figure 1.
Figure 1. A flow chart depicting the overall study design. In total, 125 men and women were enrolled
into the research study (60 men, 65 women). Following enrollment and screening for
inclusion/exclusion criteria, participants consumed their randomly assigned supplement for four
weeks. daily. Pre- and post-assessments of cardiovascular function (resting heart rate, blood pressure,
and heart function via electrocardiogram) and blood collection for the analysis of complete blood
count, comprehensive metabolic panel, and lipid panel were included. minP = minutes post first dose
of randomly assigned supplement; DYM = Dynamine
®
;
TCR = Teacrine
®
; mg = milligram.
2.2. Participants
Healthy weight to obese (not severely obese) men and women, 18–55 years, whom reported
activity levels ranging from sedentary to recreationally active, were recruited for the study (no elite
athletes) via flyers and word of mouth. Participants who reported a history of arrhythmias, a family
history of sudden cardiac deaths in immediate family members (before the age of 55 for male, before
the age of 65 for female relatives), and sensitivities to caffeine (anxiousness, nausea) were not
enrolled. Participants diagnosed with or currently being treated for bacterial infections were
excluded. Furthermore, participants were excluded if they were previously diagnosed with
cardiovascular, endocrine/metabolic, gastrointestinal, renal, pulmonary, orthopedic, immunological,
physiological, or musculoskeletal disorders. Individuals consuming more than 400 mg of caffeine per
day, current smokers, or individuals who smoked within the last three years, as well as persons with
known conditions or taking medications that may interfere with the absorption, distribution,
metabolism, or excretion of purine alkaloids, also did not partake. Anti-inflammatory agents,
including corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDS), were also not
permitted. Furthermore, any participant that had a positive, verbal pre-study drug screening for the
use of alcohol, tetrahydrocannabinol (THC)/cannabinoids, amphetamines, benzodiazepines, cocaine,
Nutrients 2020, 12, 654 4 of 20
opioids, phencyclidine, barbiturates, and cotinine was excluded. Any participant who missed three
or more doses during the four-week supplementation period was considered non-compliant and was
excluded from the analyses. Following an explanation of all study procedures risks and benefits, 161
men and women signed an KSU Institutional Review Board approved consent form to complete the
study (#18-223). Out of all enrolled participants, 125 men (n = 60) and women (n = 65) completed the
study (23.0 ± 3.3 years, 169.7 ± 8.9 cm, 72.1 ± 13.7 kg, 25.7% ± 8.4% body fat) (Table 1). Participant
reasons for not completing the study were as follows: (1) participants were classified as hypertensive
(stage two hypertension) per the 2017 American Heart Association Guidelines at Visit 1 (n = 8); (2)
participants presented with an ECG abnormality at Visit 1 (n = 7); (3) participants missed three or
more days of supplementation and were asked to not return for Visit 3 (n = 11); (4) participants did
not return for Visit 3 due to schedule conflicts (n = 5); (5) participants self-reported illness during the
supplementation period (n = 3; self-reported flu-like symptoms including chills, sweats, muscle
aches); (5) participants began supplementation and stopped responding to follow-up contact for Visit
3 (n = 2). Men and women completing the study were equally distributed within each of the five
groups (12 men, 13 women, n = 25 per group). All participants were asked to maintain their habitual
dietary intake (i.e., make no changes other than start taking their assigned supplement when
instructed) and exercise habits (i.e., if they started the study reporting sedentary, they were asked to
maintain that level of activity; if they started the study recreationally active, they were asked to
maintain that level of activity) for the duration of the study. Following study completion, participants
were compensated with a gift card.
Table 1. Baseline participant descriptions. Values are means ± SD. M—male; F—female.
Sample
Size Sex Age
(year)
Height
(cm)
Body
Mass
(kg)
Body
Fat
(%)
Resting
Heart Rate
(bpm)
Resting Systolic
Blood Pressure
(mmHg)
Resting Diastolic
Blood Pressure
(mmHg)
100 mg DYM
12 M
21.7 ±
1.3
174.5 ±
7.8
78.2 ±
11.1
16.7 ±
6.3 63.3 ± 7.5 123.0 ± 7.0 75.8 ± 8.4
13 F
23.4 ±
2.7
162.6 ±
4.4
62.7 ±
10.9
30.6 ±
6.7 66.0 ± 11.3 110.2 ± 3.8 67.0 ± 7.6
100 mg DYM +
50 mg TCR
12 M
23.5 ±
3.0
175.1 ±
6.7
83.3 ±
10.3
24.2 ±
7.0 66.9 ± 10.9 120.0 ± 6.4 74.1 ± 10.2
13 F
24.0 ±
6.5
163.3 ±
5.1
64.4 ±
14.1
31.7 ±
7.5 61.3 ± 7.6 107.5 ± 4.9 64.6 ± 5.2
150 mg DYM +25
mg TCR
12 M
24.2 ±
3.3
175.7 ±
3.5
78.1 ±
5.6
19.5 ±
2.9 63.6 ± 8.8 117.6 ± 5.8 71.5 ± 9.1
13 F
23.1 ±
2.6
164.4 ±
5.1
62.7 ±
8.0
30.7 ±
7.1 73.8 ± 10.1 111.0 ± 6.8 71.0 ± 5.4
150 mg DYM
12 M
22.8 ±
2.6
178.3 ±
7.3
80.6 ±
8.2
19.8 ±
5.6 61.1 ± 10.7 116.3 ± 7.8 74.4 ± 8.7
13 F
21.8 ±
1.8
162.7 ±
8.6
63.0 ±
15.0
27.7 ±
9.4 73.7 ± 11.2 111.2 ± 8.0 68.1 ± 6.1
Placebo
(maltodextrin)
12 M
22.8 ±
3.3
117.2 ±
7.7
82.5 ±
14.0
23.8 ±
7.8 66.9 ± 3.9 117.7 ± 7.1 76.8 ± 7.0
13 F
22.9 ±
3.8
166.5 ±
4.7
70.9 ±
11.9
31.1 ±
6.9 68.8 ± 10.1 110.0 ± 5.3 67.3 ± 7.6
2.3. Visits Overview
2.3.1. Visit 1: Pre-participation screening
Prior to starting the experimental portion of the study, all participants visited the laboratory for
pre-participation screening (Visit 1). Each individual was firstly given an overview of the
investigation, then provided their written informed consent to participate, and finally completed
medical, exercise, and dietary questionnaires. Following completion of study documents,
participants were asked to rest for 5 min in the seated position before completing resting heart rate
and blood pressure assessments (two measurements separated by 2 min). If the two measurements
varied by more than ±2 mmHg, a third measurement was taken and the average of the closest two
Nutrients 2020, 12, 654 5 of 20
values was used for later analysis. Subsequently, height and body mass were collected and then
participants were prepared for and completed baseline, resting ECG assessment by a trained member
of the research team. The ECG printout was reviewed by a medical doctor or clinical exercise
physiologist for abnormalities. Qualifying participants were scheduled for Visit 2.
2.3.2. Visits 2 and 3
Visit 2 began with assessments of body mass and composition (dual energy X-ray
absorptiometry (DXA)), followed by blood collection, resting blood pressure, and heart rate, and
lastly, a resting ECG. A blinded research team member then randomly assigned the individual to a
study group. Participants then consumed their first dose and remain in the laboratory for 2 h. Resting
heart rate and blood pressure were assessed every 30 min post (minP) first dose supplementation (30
minP, 60 minP, 90 minP, and 120 minP), while resting ECG was assessed at 60 minP only. Following
the 120-minP time point, participants were given a bottle of their assigned supplement (coded for
double-blind administration), instructed on how to consume the supplement once daily, and
scheduled to return ( ±2 h time frame from Visit 2) to the laboratory four weeks later for Visit 3. Visit
3 procedures occurred in the same manner as Visit 2, excluding the height assessment and DXA scan.
2.4. Laboratory Assessments
2.4.1. Anthropometric Measurements
Body mass was measured at the beginning of each laboratory visit. Height was measured
following enrollment into the investigation during the first visit only. Participants completed all mass
and height measurements without shoes using the same calibrated stadiometer and scale (Tanita WB
3000, Arlington Heights, IL). For demographic purposes, participant’s baseline body fat percentage
was assessed via a full body DXA scan (Lunar iDXA, General Electric, Chicago, IL). The DXA scanner
was calibrated according to manufacturer guidelines and the positioning of the participant was
conducted according to manufacturer recommendations. All assessments occurred in the fasted state
(≥8 h).
2.4.2. Blood sampling and analysis
Participant’s fasting venous blood was collected during Visits 2 and 3 from a vein in the
antecubital space by a research team member trained in phlebotomy. Blood was collected into serum
separator (SST) and ethylenediaminetetraacetic acid (EDTA) treated Vacutainer® tubes. SST tubes
were allowed to clot for 10 min prior to centrifugation at room temperature for 15 min (Model #642E,
Drucker Diagnostics, Port Matila, PA, USA). EDTA whole-blood samples were inverted 8–10 times
immediately after collection. Whole blood and serum were analyzed by a third-party laboratory
(Laboratory Corporation of America™) for CBC with differential, CMP, and lipids. The CBC with
differential allowed for the quantification of white blood cells, red blood cells, hemoglobin,
hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin
concentration, red cell distribution width, platelets, neutrophils, lymphocytes, monocytes,
eosinophils, basophils, absolute neutrophils (derived by multiplying the white blood cell count
(WBC) by the percent of neutrophils in the differential WBC count; the percentage of neutrophils
consists of the segmented/fully mature neutrophils plus the bands/almost mature neutrophils),
absolute lymphocytes (absolute lymphocyte count = WBC times lymphocytes divided by 100),
absolute monocytes (absolute monocyte count = WBC times percent monocytes times 100), absolute
eosinophils (absolute eosinophils = WBC times eosinophils divided by 100), absolute basophils
(calculated by multiplying the percentage of basophils by the total number of WBC), and immature
granulocytes and absolute immature granulocytes (metamyelocytes, myelocytes; no bands or blast
cells). The CMP allowed for the assessment of glucose, blood urea nitrogen, creatinine, estimated
glomerular filtration rate, blood urea nitrogen–creatinine ratio, sodium, potassium, chloride, total
carbon dioxide, calcium, total protein, albumin (A), total globulin (G), A/G ratio, total bilirubin,
alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase. The lipid panel
Nutrients 2020, 12, 654 6 of 20
assessment consisted of total cholesterol, triglycerides, high-density lipoproteins, very-low density
lipoproteins, and low-density lipoproteins.
2.4.3. Resting heart rate and blood pressure
Resting heart rate(s) and blood pressure(s) were collected during all three visits. Prior to the
measurements of heart rate and blood pressure, participants were fitted with a heart rate monitor
(T31 Coded Transmitter and Polar Pt1 Heart Rate Monitor, Polar® Electro Inc., Bethpage, NY) and
asked to sit on a chair for five minutes with back support to allow the blood pressure to adjust to
resting conditions. Systolic and diastolic blood pressures were assessed by trained members of the
research team, manually, using a sphygmomanometer (Diagnostix™ 788, American Diagnostic
Corporation, Hauppauge, NY, USA), cuff of the appropriate size (Adcuff™, American Diagnostic
Corporation, Hauppauge, NY, USA), and a stethoscope (Classic II, 3M™ Littmann®, St. Paul, MN,
USA). During PRE assessments (i.e. before visit supplementation), blood pressure was measured
twice with a 2-min interval between the measurements and the average score of the two measures
being recorded nearest to one mmHg. During 30-minP, 60-minP, 90-minP, and 120-minP
assessments, only a single blood pressure was collected. Blood pressure was always collected in the
same arm of each participant.
2.4.4. Resting Electrocardiogram
Resting ECG(s) were collected during all three visits. A 12-lead ECG was conducted (I–III, V1–
V6, aVR, aVL, aVF) following proper skin prep procedures. All females were asked to wear a bra
with no metal. Participants were asked to remain in the seated position with their feet flat on the
ground for 10 min. Cardiac rhythm was monitored and recorded via a GE Case® 8000 from minutes
5–10. Data were printed from the 12-lead ECG machine and PR interval, QRS duration, corrected
QTc, P axis, R axis, T axis, P duration, RR interval, and PP interval were recorded.
Reliability statistics were calculated from the pre-ECG recordings collected on the first two visits
(i.e., prior to any supplementation) from a random sample of 10 men (23.9 ± 3.9 years; 178 ± 6 cm; 82.8
± 10.5 kg; body mass index = 26 ± 2.2) and 10 women (24.4 ± 7.1 years; 164 ± 5 cm; 66.6 ± 19.1 kg; BMI
= 25.9 ± 4.9) that were drawn from the entire sample with each group being represented. After
confirming that no statistical differences existed between time points, intraclass correlation
coefficients (ICC3,1) and standard error of the measurement (SEM) were calculated for PR interval
(ICC3,1 = 0.85, SEM = 8.63 ms), QRS duration (ICC3,1 = 0.92, SEM = 3.52 ms), QTC (ICC3,1 = 0.74, SEM =
16.9 ms), P axis (ICC3,1 = 0.84, SEM = 8.68°), R axis (ICC3,1 = 0.91, SEM = 7.00°), T axis (ICC3,1 = 0.79, SEM
= 8.81°), P duration (ICC3,1 = 0.9, SEM = 4.84 ms), RR interval (ICC3,1 = 0.84, SEM = 96.8 ms), and PP
interval (ICC3,1 = 0.9, SEM = 92.0 ms).
2.4.5. Supplementation intervention
Participants were matched according to sex and then randomly assigned to one of five groups:
low-dose DYM (100 mg), high-dose DYM (150 mg), low-dose DYM with TCR (100 mg + 50 mg), high-
dose DYM with TCR (150 mg + 25 mg), and 125 mg of maltodextrin (placebo). The terms high- and
low-dose are terms used specifically to differentiate between groups in this manuscript and should
not be deemed as definitive “high” and “low” dosages for the given supplements. Participants were
asked to take their supplement in the morning with water (~12 fluid ounces) and food. Supplement
compliance was monitored by asking the participant to return their pill bottle with any pills missed
upon arrival to a member of the research team. Research team members also verbally confirmed the
number of pills missed with each participant to account for supplement compliance. All supplements
were provided by Compound Solutions, Inc. Supplements were indistinguishable to participants, as
the pills were the same color, shape, and texture. Supplements were distributed by members of the
research team who were blinded to treatments. The supplement key for each group was kept by a
member of the research team not involved in data collection.
Nutrients 2020, 12, 654 7 of 20
2.4.6. Diet Tracking
During the four-week trial, participants were asked to track their diet two weekdays and one
weekend day, for a total of three days per week. Participants either recorded all food and drinks on
a hard-copy diet log or into MyFitnessPal (Under Armour, Inc, Baltimore, MD). A single member of
the research team entered any hard-copy diet logs into MyFitnessPal. Carbohydrate, fat, protein, and
total calories were assessed from week one and week four to determine if participants maintained
their normal diets. Dietary intakes for macronutrients and total calories are displayed in Table 8.
2.5. Statistical Analysis
A priori analysis using a moderate effect size f (0.25), standard alpha (0.05), and maximum beta
(0.95) for a five-group, four-time-point repeated measures design, statistical software (G*Power,
v.3.1.9.4, Universitat Dusseldorf, Germany) indicated that a minimum of 60 total participants were
necessary to observe statistical differences. Prior to group comparisons, the assumption of normality
was verified by the Shapiro–Wilk test. Subsequently, separate three-way (group × sex × time) analyses
of variance with repeated measures were performed on all measures of cardiovascular function and
blood biomarkers. The Greenhouse–Geisser adjustment was applied when the assumption of
sphericity was violated. Following any significant F-ratio, pairwise comparisons were performed
using the Bonferonni post hoc analysis. The magnitude of significant differences was assessed by
effect sizes (i.e., eta squared, ɳ2) and qualitatively described as small (0.2–0.49), moderate (0.5–079),
or large (≥ 0.80) [31]. Dependent t-tests were used to determine differences in diet from week one to
week four. Criterion alpha was set at p ≤ 0.05. All statistical analyses were performed using JASP
0.10.2 (Amsterdam, the Netherlands). All data are reported as means ± standard deviation.
3. Results
3.1. Cardiovascular Function
Regardless of group and sex, significant main effects for time were noted for heart rate (F = 6.87,
p < 0.001, ɳ2 = 0.02), systolic blood pressure (F = 6.75, p < 0.001, ɳ2 = 0.02), QRS duration (F = 2.73, p =
0.035, ɳ2 < 0.01), QTc (F = 6.31, p < 0.001, ɳ2 = 0.01), P axis (F = 4.77, p = 0.002, ɳ2 = 0.01), RR interval (F =
28.84, p < 0.001, ɳ2 = 0.06), and PP interval (F = 15.43, p < 0.001, ɳ2 = 0.03). Compared to their respective
values at PRE, heart rate was decreased at 60 minP on Visit 2 (p = 0.018) and Visit 3 (p < 0.001), while
RR interval and PP interval were both elevated (p < 0.001) at 60 minP on Visit 2 and Visit 3; the RR
interval was also elevated from Visit 1 at PRE on Visit 2 (p = 0.010). QTC (p = 0.013) and P axis (p =
0.010) were depressed from PRE on Visit 2 at 60 minP only. A trend for decreased systolic blood
pressure was noted on Visit 3 PRE compared to Visit 1 (p = 0.082). However, neither sex nor group
affected these responses. Data collapsed across groups for changes in heart rate and blood pressure
are illustrated in Figure 2, while collapsed data derived from the electrocardiogram are presented in
Table 2.
Nutrients 2020, 12, 654 8 of 20
Figure 2. Collapsed data (n = 125; group and sex) for group changes in (A) heart rate and (B) blood
pressure (systolic (toward the top) and diastolic (toward the bottom)).
* Significantly (p < 0.05) different
from respective visit's PRE; # Different (p < 0.10) from Visit 1 (V1); minP = minutes post PRE; mmHg = millimeters
of mercury; bpm = beats per minute.
Table 2. Collapsed (n = 125; group and sex) data for group changes in electrocardiogram measures. n
= 25 participants per group.
Visit 1 Visit 2 Visit 3
PRE PRE 60 minP PRE 60 minP
PR Interval (ms) 145 ± 17 146 ± 17 147 ± 17 145 ± 16 149 ± 34
QRS Duration (ms) 88.7 ± 9 89.7 ± 9 89.8 ± 8.8 89.6 ± 9.1 89.2 ± 8.9
QT
C
(ms) 412 ± 23 410 ± 23 406 ± 24 * 412 ± 23 409 ± 24
P Axis (◦) 56.4 ± 19.1 56.5 ± 19.1 51.9 ± 22.3 * 53.9 ± 20.9 51.5 ± 22.2
R Axis (◦) 75.7 ± 15.3 75.5 ± 17.3 74.1 ± 17.5 75.4 ± 17.4 75.1 ± 17.4
T Axis (◦) 51.8 ± 14 53 ± 14.3 53.6 ± 17.6 52.6 ± 13.6 52.4 ± 13.3
P Duration (ms) 94.5 ± 10.2 94.2 ± 10.1 93.3 ± 11.3 93.4 ± 10 94.4 ± 10.2
RR Interval (ms) 928 ± 158 961 ± 157 # 1030 ± 166 * 950 ± 185 1024 ± 176 *
PP Interval (ms) 935 ± 214 959 ± 161 1022 ± 173 * 948 ± 188 1010 ± 167 *
Nutrients 2020, 12, 654 9 of 20
* Significantly (p < 0.05) different from PRE on respective visit. # Significantly (p < 0.05) different from
Visit 1. minP = minutes post PRE; PR Interval = time from the onset of the P wave to the start of the
QRS complex; QRS duration = ventricular depolarization; QT = time duration between the onset of
the QRS complex and the end of the T wave (ventricular depolarization to complete repolarization);
c = corrected for heart rate; P duration = time of P wave (atrial depolarization); RR interval = time
elapsed between successive R waves; PP interval = time elapsed between successive P waves; axis
represents overall electrical activity of the heart.
Although a group × sex × time interaction was observed for P duration (F = 1.72, p = 0.044, ɳ2 =
0.02), the observed differences were between sexes at different time points. Specifically, P duration
for women consuming 100 mg of Dynamine® was less on Visit 1 (p = 0.028) and at 60 minP on Visit 2
(p = 0.033) compared to men consuming 100 mg of DYM + 50 mg of TCR at 60 minP on Visit 3.
Likewise, that for women consuming 150 mg of DYM was less at 60 minP on Visit 2 compared to men
consuming 100 mg of DYM + 50 mg of TCR at 60 minP on Visit 3 (p = 0.021). Otherwise, no differences
were noted between sexes and groups in the P duration response compared to PRE values.
3.2. Blood Biomarkers
Regardless of group and sex, significant main effects for time were observed for markers
analyzed within the complete blood count. Specifically, increased mean corpuscular volume (F =
12.53, p < 0.001, ɳ2 = 0.01), mean corpuscular hemoglobin (F = 5.81, p = 0.018, ɳ2 < 0.01), basophils (F =
7.93, p = 0.006, ɳ2 = 0.03), and absolute eosinophils (F = 6.98, p = 0.010, ɳ2 = 0.01) were found on Visit 3.
Although a group × time interaction was observed for platelets (F = 2.75, p = 0.032, ɳ2 = 0.01), post hoc
analysis did not reveal any significant group differences. Group × sex × time interactions were also
observed for mean corpuscular hemoglobin concentration (F = 3.15, p = 0.017, ɳ2 = 0.02), red cell
distribution width (F = 2.53, p = 0.045, ɳ2 = 0.01), and absolute lymphocytes (F = 2.89, p = 0.026, ɳ2 =
0.02). However, post hoc analysis only revealed specific differences in mean corpuscular hemoglobin
concentrations where values in men consuming the placebo (34.1 ± 0.9 g/dL) were greater than those
found in women consuming 100 mg of DYM (32.9 ± 0.8 g/dL, p = 0.028) and 150 mg of DYM + 25 mg
of TCR (32.8 ± 1.0 g/dL, p = 0.011) on Visit 3. No other specific differences were observed. Group and
collapsed complete blood count comparisons between Visits 2 and 3 are presented in Table 3 and
Table 4.
Regardless of group and sex, significant main time effects were observed for markers analyzed
within the complete metabolic panel. Specifically, increased creatinine (F = 8.91, p = 0.004, ɳ2 < 0.01)
and decreased estimated glomerular filtration rate (F = 4.48, p = 0.037, ɳ2 < 0.01), chloride (F = 4.87, p
= 0.030, ɳ2 = 0.01), carbon dioxide (F = 5.29, p = 0.023, ɳ2 = 0.01), bilirubin (F = 5.03, p = 0.027, ɳ2 < 0.01),
and alanine aminotransferase (F = 4.21, p = 0.043, ɳ2 < 0.01) were noted on Visit 3. Additionally, group
x time interactions were seen for blood urea nitrogen (F = 3.30, p = 0.014, ɳ2 = 0.01), total globulins (F
= 3.34, p = 0.013, ɳ2 = 0.013), alanine aminotransferase (F = 2.82, p = 0.028, ɳ2 < 0.01), and total proteins
(F = 2.61, p = 0.040, ɳ2 = 0.01), but post hoc analysis only revealed that blood urea nitrogen was greater
(p = 0.014) in participants consuming 100 mg of DYM than those consuming 100 mg of DYM + 50 mg
of TCR on Vi sit 3 . No oth er di ffer enc es we re o bserved. Group and collapsed complete metabolic panel
comparisons between Visits 2 and 3 are presented in Table 5 and Table 6.
Regardless of group and sex, a significant main effect was found for increased high-density
lipoproteins (F = 10.10, p = 0.002, ɳ2 < 0.01) on Visit 3. Group × time interactions were also seen for
triglycerides (F = 2.65, p = 0.037, ɳ2 = 0.01) and high-density lipoproteins (F = 2.48, p = 0.048, ɳ2 < 0.01),
but post hoc analyses revealed only a trend for increased (p = 0.052) high-density lipoproteins for
participants consuming 150 mg of Dynamine® on Visit 3 compared to Visit 2. No other differences
were observed. Group and collapsed lipid panel comparisons between Visits 2 and 3 are presented
in Table 7.
Nutrients 2020, 12, 654 10 of 20
Table 3. Group (n = 25 per group) and average complete blood count comparisons between Visits 2 and 3.
100 mg of DYM 100 mg of DYM + 50 mg of TCR 150 mg of DYM + 25 mg of TCR 150 mg of DYM Placebo Average
White Blood Cells
(× 103/µL)
Visit 2 5.37 ± 1.26 6.20 ± 1.62 5.78 ± 1.26 5.76 ± 1.39 5.50 ± 1.58 5.71 ± 1.43
Visit 3 5.68 ± 1.62 5.92 ± 1.97 5.71 ± 1.28 5.56 ± 1.30 5.53 ± 1.29 5.68 ± 1.49
Red Blood Cells
(× 106/µL)
Visit 2 4.83 ± 0.41 4.84 ± 0.41 4.64 ± 0.40 4.65 ± 0.33 4.83 ± 0.57 4.76 ± 0.43
Visit 3 4.83 ± 0.39 4.82 ± 0.47 4.61 ± 0.40 4.67 ± 0.26 4.84 ± 0.53 4.76 ± 0.42
Hemoglobin
(g/dL)
Visit 2 14.6 ± 1.0 14.4 ± 1.0 13.6 ± 1.0 14.1 ± 0.9 14.2 ± 1.2 14.1 ± 1.0
Visit 3 14.6 ± 1.0 14.3 ± 1.1 13.7 ± 1.1 19.5 ± 25.1 14.2 ± 1.2 15.2 ± 11.1
Hematocrit
(%)
Visit 2 43.4 ± 2.9 43.7 ± 11.7 40.3 ± 2.2 42 ± 3 42.3 ± 3.4 42.3 ± 5.6
Visit 3 44.1 ± 2.9 42.7 ± 3.0 40.9 ± 2.6 41.4 ± 3.4 42.7 ± 3.1 42.3 ± 3.2
Mean Corpuscular Volume
(fL)
Visit 2 89.7 ± 3.6 88.5 ± 3.6 88.4 ± 4.8 88.7 ± 4.7 88.0 ± 5.0 88.6 ± 4.4
Visit 3 91.0 ± 3.6 88.9 ± 3.2 88.8 ± 4.3 89.3 ± 5.5 88.6 ± 6.2 89.3 ± 4.7*
Mean Corpuscular Hemoglobin
(pg)
Visit 2 29.9 ± 1.5 29.7 ± 1.0 29.9 ± 1.8 30.1 ± 1.9 29.4 ± 1.8 29.8 ± 1.6
Visit 3 30.2 ± 1.1 29.7 ± 1.1 30.1 ± 1.6 30.5 ± 1.9 29.6 ± 1.9 30.0 ± 1.6 *
Mean Corpuscular Hemoglobin Concentration
(g/dL)
Visit 2 33.4 ± 0.8 33.6 ± 0.8 33.3 ± 1.1 33.5 ± 0.8 33.6 ± 0.9 33.5 ± 0.9
Visit 3 33.0 ± 0.8 33.5 ± 0.5 33.4 ± 1.1 33.4 ± 0.9 33.7 ± 0.9 33.4 ± 0.9
Red Cell Distribution Width
(%)
Visit 2 13.4 ± 0.7 13.2 ± 0.5 13.2 ± 0.9 13.2 ± 0.6 13.4 ± 0.6 13.3 ± 0.6
Visit 3 13.3 ± 0.5 13.3 ± 0.5 13.3 ± 0.7 13.3 ± 0.6 13.4 ± 0.6 13.3 ± 0.6
Platelets
(× 103/µL)
Visit 2 236 ± 55 265 ± 48 254 ± 42 240 ± 49 255 ± 55 250 ± 50
Visit 3 220 ± 60 246 ± 57 256 ± 41 247 ± 50 266 ± 61 247 ± 56
* Significantly (p < 0.05) different from Visit 2
Nutrients 2020, 12, 654 11 of 20
Table 4. Group (n = 25 per group) and average complete blood count comparisons between Visits 2 and 3.
100 mg of DYM 100 mg of DYM + 50 mg of TCR 150 mg of DYM + 25 mg of TCR 150 mg of DYM Placebo Average
Absolute Neutrophils
(× 103/µL)
Visit 2 2.68 ± 1.04 3.06 ± 1.47 2.82 ± 0.85 2.77 ± 1.24 2.56 ± 1.30 2.77 ± 1.18
Visit 3 2.78 ± 1.28 2.85 ± 1.58 2.82 ± 0.85 2.58 ± 1.12 2.60 ± 0.90 2.72 ± 1.15
Absolute Lymphocytes
(× 103/µL)
Visit 2 2.09 ± 0.33 2.31 ± 0.44 2.30 ± 0.50 2.13 ± 0.51 2.07 ± 0.51 2.18 ± 0.47
Visit 3 2.25 ± 0.50 2.31 ± 0.72 2.24 ± 0.51 2.20 ± 0.48 2.20 ± 0.60 2.24 ± 0.56
Absolute Monocytes
(× 103/µL)
Visit 2 0.463 ± 0.124 0.519 ± 0.172 0.432 ± 0.149 0.396 ± 0.112 0.475 ± 0.165 0.455 ± 0.149
Visit 3 0.445 ± 0.122 0.491 ± 0.216 0.470 ± 0.122 0.414 ± 0.159 0.467 ± 0.134 0.458 ± 0.153
Absolute Eosinophils
(× 103/µL)
Visit 2 0.183 ± 0.169 0.162 ± 0.102 0.144 ± 0.112 0.138 ± 0.077 0.175 ± 0.099 0.160 ± 0.116
Visit 3 0.186 ± 0.146 0.191 ± 0.177 0.157 ± 0.112 0.181 ± 0.112 0.192 ± 0.132 0.181 ± 0.136 *
Absolute Basophils
(× 103/µL)
Visit 2 0.017 ± 0.038 0.014 ± 0.036 0.012 ± 0.033 <0.010 <0.010 0.008 ± 0.028
Visit 3 0.009 ± 0.029 0.009 ± 0.029 0.027 ± 0.046 0.014 ± 0.036 0.008 ± 0.028 0.014 ± 0.034
Immature Granulocytes
(× 103/µL)
Visit 2 0.208 ± 0.415 0.429 ± 0.978 0.080 ± 0.277 0.261 ± 0.689 0.333 ± 0.637 0.256 ± 0.632
Visit 3 0.318 ± 0.477 0.364 ± 0.79 <0.010 0.048 ± 0.218 0.208 ± 0.658 0.188 ± 0.529
Absolute Immature Granulocytes
(× 103/µL)
Visit 2 0.017 ± 0.038 0.029 ± 0.072 0.004 ± 0.020 0.009 ± 0.029 0.025 ± 0.044 0.016 ± 0.043
Visit 3 0.014 ± 0.035 0.014 ± 0.035 <0.010 0.010 ± 0.030 0.013 ± 0.034 0.010 ± 0.030
* Significantly (p < 0.05) different from Visit 2.
Nutrients 2020, 12, 654 12 of 20
Table 5. Group (n = 25 per group) and average complete metabolic panel comparisons between Visits 2 and 3.
100 mg of DYM 100 mg of DYM + 50 mg of TCR 150 mg of DYM + 25 mg of TCR 150 mg of DYM Placebo Average
Glucose
(mg/dL)
Visit 2 87.5 ± 7.2 89.1 ± 7.4 90.2 ± 5.2 91.2 ± 9.4 91.0 ± 8.5 89.8 ± 7.7
Visit 3 87.2 ± 8.5 88.2 ± 9.1 88.9 ± 6.4 91.9 ± 7.6 89.9 ± 8.8 89.2 ± 8.2
Blood Urea Nitrogen
(mg/dL)
Visit 2 15.2 ± 4.1 14.0 ± 4.0 14.0 ± 4.6 14.2 ± 4.1 14.0 ± 3.3 14.3 ± 4.0
Visit 3 16.5 ± 4.7 12.6 ± 3.7# 14.3 ± 4.9 14.0 ± 3.9 14.1 ± 2.8 14.3 ± 4.2
Creatinine
(mg/dL)
Visit 2 0.984 ± 0.220 0.955 ± 0.161 0.870 ± 0.191 0.947 ± 0.246 0.908 ± 0.172 0.932 ± 0.201
Visit 3 1.003 ± 0.210 0.999 ± 0.177 0.889 ± 0.198 0.981 ± 0.230 0.914 ± 0.172 0.957 ± 0.200*
Estimated Glomerular Filtration Rate
(mL/min/1.73)
Visit 2 96.6 ± 17.8 94.1 ± 26.3 107.8 ± 16.7 93.3 ± 29.0 104.0 ± 14.9 99.3 ± 22.0
Visit 3 84.1 ± 31.0 92.0 ± 15.4 105.7 ± 15.4 91.6 ± 26.7 103.8 ± 16 95.5 ± 22.9*
Blood Urea Nitrogen–Creatinine Ratio Visit 2 16.1 ± 5.0 14.8 ± 4.4 16.0 ± 4.2 14.9 ± 5.4 15.3 ± 3.3 15.4 ± 4.5
Visit 3 16.7 ± 4.2 12.9 ± 4.1 16.0 ± 4.0 14.7 ± 4.1 15.5 ± 3.1 15.1 ± 4.1
Sodium
(mmol/L)
Visit 2 141 ± 2 140 ± 2 140 ± 2 141 ± 2 140 ± 2 141 ± 2
Visit 3 141 ± 1 140 ± 2 140 ± 2 141 ± 2 141 ± 2 141 ± 2
Potassium
(mmol/L)
Visit 2 4.80 ± 0.42 4.62 ± 0.48 4.46 ± 0.44 4.98 ± 1.91 4.35 ± 0.32 4.64 ± 0.95
Visit 3 4.61 ± 0.52 4.63 ± 0.83 4.41 ± 0.29 4.64 ± 0.70 4.60 ± 0.42 4.58 ± 0.59
Chloride
(mmol/L)
Visit 2 102 ± 3 101 ± 4 102 ± 2 103 ± 2 102 ± 2 102 ± 3
Visit 3 103 ± 2 103 ± 2 102 ± 2 103 ± 3 103 ± 2 103 ± 2 *
Total Carbon Dioxide
(mmol/L)
Visit 2 24.3 ± 2.3 24.3 ± 2.0 23.2 ± 2.5 23.4 ± 2.2 23.9 ± 2.2 23.8 ± 2.2
Visit 3 23.2 ± 2.1 23.3 ± 1.7 22.2 ± 2.3 23.2 ± 2.6 24.3 ± 2.3 23.3 ± 2.3 *
Calcium
(mg/dL)
Visit 2 9.64 ± 0.27 9.53 ± 0.31 9.47 ± 0.37 9.32 ± 0.45 9.53 ± 0.34 9.50 ± 0.37
Visit 3 16.77 ± 23.90 9.40 ± 0.34 9.44 ± 0.29 9.44 ± 0.33 9.50 ± 0.38 10.87 ± 10.77
* Significantly (p < 0.05) different from Visit 2. # Significantly (p < 0.05) different from 100 mg of Dynamine on Visit 3.
Nutrients 2020, 12, 654 13 of 20
Table 6. Group (n = 25 per group) and average complete metabolic panel comparisons between Visits 2 and 3.
100 mg of DYM 100 mg of DYM + 50 mg of TCR 150 mg of DYM + 25 mg of TCR 150 mg of DYM Placebo Average
Total Protein
(g/dL)
Visit 2 7.18 ± 0.34 7.26 ± 0.52 7.06 ± 0.53 6.92 ± 0.72 7.18 ± 0.25 7.12 ± 0.51
Visit 3 7.07 ± 0.31 7.17 ± 0.51 7.08 ± 0.36 7.11 ± 0.76 7.18 ± 0.39 7.12 ± 0.49
Albumin (A)
(g/dL)
Visit 2 4.67 ± 0.26 4.61 ± 0.33 4.60 ± 0.42 4.56 ± 0.30 4.66 ± 0.32 4.62 ± 0.33
Visit 3 4.65 ± 0.26 4.53 ± 0.31 4.54 ± 0.38 4.62 ± 0.42 4.64 ± 0.31 4.59 ± 0.34
Total Globulin (G)
(g/dL)
Visit 2 2.47 ± 0.19 2.65 ± 0.40 2.58 ± 0.43 2.59 ± 0.42 2.58 ± 0.35 2.57 ± 0.37
Visit 3 2.43 ± 0.23 2.52 ± 0.42 2.67 ± 0.38 2.67 ± 0.38 2.58 ± 0.36 2.57 ± 0.37
A/G Ratio Visit 2 1.90 ± 0.19 1.78 ± 0.31 1.83 ± 0.35 1.81 ± 0.31 1.84 ± 0.32 1.83 ± 0.30
Visit 3 1.94 ± 0.24 1.84 ± 0.3 1.73 ± 0.28 1.76 ± 0.31 1.84 ± 0.29 1.82 ± 0.29
Bilirubin, Total
(mg/dL)
Visit 2 0.529 ± 0.229 0.496 ± 0.272 0.504 ± 0.348 0.424 ± 0.230 0.496 ± 0.321 0.489 ± 0.282
Visit 3 0.448 ± 0.250 0.468 ± 0.281 0.483 ± 0.248 0.391 ± 0.095 0.454 ± 0.213 0.449 ± 0.226 *
Alkaline Phosphatase
(IU/L)
Visit 2 61.8 ± 15.6 66.9 ± 23.1 66.4 ± 21.3 62.5 ± 15.8 68.4 ± 18.2 65.2 ± 18.9
Visit 3 60.3 ± 15.5 65.5 ± 20.8 67.6 ± 21.2 62.8 ± 15.6 71.4 ± 24.4 65.6 ± 19.9
Aspartate Aminotransferase
(IU/L)
Visit 2 30.9 ± 22.8 23.4 ± 10.0 21.7 ± 7.2 23.2 ± 8.2 24.6 ± 8.9 24.7 ± 12.9
Visit 3 28.9 ± 17.7 24.7 ± 10.1 23.2 ± 8.9 24.6 ± 11.6 22.0 ± 7.1 24.7 ± 11.6
Alanine Aminotransferase
(IU/L)
Visit 2 23.8 ± 11.7 22.5 ± 22.7 19.2 ± 9.7 14.8 ± 6.4 21.3 ± 10.6 20.3 ± 13.6
Visit 3 19.6 ± 7.9 22.4 ± 18.0 19.7 ± 10 15.7 ± 6.7 18.8 ± 9.6 19.3 ± 11.3 *
* Significantly (p < 0.05) different from Visit 2. # Significantly (p < 0.05) different from 100 mg of Dynamine on Visit 3.
Nutrients 2020, 12, 654 14 of 20
Table 7. Group (n = 25 per group) and average lipid panel comparisons between Visits 2 and 3.
100 mg of DYM 100 mg of DYM + 50 mg of TCR 150 mg of DYM + 25 mg of TCR 150 mg of DYM Placebo Average
Total Cholesterol
(mg/dL)
Visit 2 158 ± 26 167 ± 30 155 ± 34 145 ± 22 175 ± 22 160 ± 28
Visit 3 160 ± 35 166 ± 24 157 ± 34 148 ± 23 167 ± 34 160 ± 31
Triglycerides
(mg/dL)
Visit 2 69.8 ± 29.7 86.3 ± 39.6 84.9 ± 32.2 84.3 ± 39.5 96.0 ± 60.6 84.3 ± 41.8
Visit 3 75.3 ± 31.9 77.8 ± 35.5 91.1 ± 41.3 83.4 ± 30.5 82.2 ± 44.1 81.9 ± 36.8
High-Density Lipoproteins
(mg/dL)
Visit 2 63.8 ± 13.9 56.7 ± 13.7 59.3 ± 12.4 54.7 ± 12.3 58.2 ± 17.4 58.5 ± 14.1
Visit 3 63.2 ± 14.0 60.0 ± 16.1 61.1 ± 15.6 59.5 ± 14.2 57.4 ± 22.2 60.2 ± 16.5 *
Very-Low-Density Lipoproteins
(mg/dL)
Visit 2 13.9 ± 6.0 17.2 ± 7.9 15.4 ± 5.5 15.8 ± 7.9 18.0 ± 11.6 16.0 ± 8.0
Visit 3 15.0 ± 6.6 15.8 ± 7.4 16.8 ± 7.8 16.3 ± 6.1 17.2 ± 9.4 16.2 ± 7.4
Low-Density Lipoproteins
(mg/dL)
Visit 2 80.8 ± 21.0 93.8 ± 23.9 80.0 ± 28.5 74.9 ± 19.7 98.6 ± 22.4 85.6 ± 24.6
Visit 3 81.6 ± 26.9 90.2 ± 24.2 79.5 ± 27.3 72.3 ± 20.1 96.4 ± 24.4 84.2 ± 25.7
* Significantly (p < 0.05) different from Visit 2.
Nutrients 2020, 12, 654 15 of 20
3.1. Dietary Tracking
No significant differences in diet were found carbohydrate, fat, protein, or total calories from
week one to week four within groups (Table 8).
Table 8. Macronutrient intakes and total calories at week one and week four for all participants. Values are
mean ± SD.
Sample
Size Sex Carbohydrates
(g) Fat (g) Protein
(g) Total Calories
p-Value
100 mg of DYM
12 M
Week 1 202 ± 43 72 ± 20 177 ± 27 2166 ± 323
0.17
Week 4 206 ± 39 64 ± 34 166 ± 34 2060 ± 311
13 F
Week 1 174 ± 52 61 ± 26 98 ± 34 1637 ± 409
0.72
Week 4 188 ± 46 63 ± 25 90 ± 25 1681 ± 356
100 mg of DYM
+ 50 mg of TCR
12 M
Week 1 231 ± 30 67 ± 20 118 ± 33 2002 ± 253
0.13
Week 4 229 ± 33 75 ± 26 126 ± 27 2091 ± 268
13 F
Week 1 181 ± 31 43 ± 12 95 ± 22 1488 ± 221
0.53
Week 4 176 ± 29 52 ± 19 89 ± 22 1528 ± 174
150 mg of DYM
+25 mg of TCR
12 M
Week 1 238 ± 57 74 ± 34 147 ± 51 2172 ± 330
0.48
Week 4 253 ± 52 70 ± 34 150 ± 62 2244 ± 517
13 F
Week 1 186 ± 79 58 ± 13 90 ± 28 1627 ± 337
0.75
Week 4 194 ± 68 54 ± 14 96 ± 22 1652 ± 307
150 mg of DYM
12 M
Week 1 198 ± 214 72 ± 24 161 ± 48 2087 ± 345
0.66
Week 4 214 ± 43 65 ± 20 149 ± 49 2037 ± 388
13 F
Week 1 166 ± 46 56 ± 13 83 ± 27 1501 ± 232
0.12
Week 4 171 ± 58 62 ± 16 89 ± 21 1600 ± 306
Placebo
(maltodextrin)
12 M
Week 1 170 ± 56 85 ± 20 158 ± 65 2078 ± 405
0.73
Week 4 185 ± 42 72 ± 23 161 ± 60 2028 ± 212
13 F
Week 1 213 ± 85 56 ± 26 89 ± 32 1711 ± 425
0.15
Week 4 186 ± 98 57 ± 21 84 ± 37 1598 ± 434
4. Discussion
To the best of our knowledge, no study previously examined human oral consumption of DYM
alone, nor its combination with TCR, and research on TCR alone in humans is somewhat limited.
Given their hypothesized similarities with caffeine and potential for reduced habituation and
physiological side effects [11,12], DYM and TCR represent intriguing neuro-energetic alternatives. In
animal models, TCR [21,32] and DYM [25] alone were determined to be safe. The results of the present
study support the hypothesis that 28 days of supplementation does not cause abnormal changes in
resting cardiovascular parameters (i.e., heart rate, blood pressure, and electrical conductivity of the
Nutrients 2020, 12, 654 16 of 20
heart) or standard hematological safety markers. These findings are in reference to a sample of
relatively healthy, young men and women (i.e., similar to the participants in the present study).
4.1. Cardiovascular Function
Resting heart rate is a clinical parameter indicative of health that is easily assessed. Normal
values are typically classified between 60 and 100 bpm, and it is not uncommon for values as low as
~30 bpm to be recorded in individuals in excellent physical condition. Moreover, it was found that
women often have higher resting heart rates than men [33]. Results from our investigation indicate
that, compared to their values at PRE for each respective visit, heart rate was decreased at 60 minP
on Visit 2 and Visit 3, regardless of group and sex. Although a negligible decrease in resting HR from
PRE was seen on Visit 2 and Visit 3, as evidenced by a less than small effect size, values similarly
remained within healthy parameters (i.e., 60–100 bpm).
To date this is the first investigation to examine the impact of DYM on resting heart rate, while
a few investigations examined TCR. Li et al. [34] examined the effects of TCR (30 mg/kg) on resting
heart rate of hypertensive and normotensive rats at baseline, as well as every 30 min up to 180 min
post TCR administration. No changes in resting heart rate were found. Likewise, TCR supplemented
at doses of 200 mg and 300 mg by male and female human subjects for eight weeks did not negatively
affect resting heart rate [17]. Similar null results were found by Ziegenfuss et al. [14], whose team
investigated the acute influence of a 200-mg dose of TCR on heart rate before, as well as 30-minP, 60-
minP, 90-minP, 120-minP, 150-minP, and 180-minP supplementation, compared to placebo.
Moreover, another study by Kuhman and colleagues [15] compared the effects of men and women’s
acute consumption (baseline, and 1 h, 2 h, 3 h, and 4 h post supplementation) of a multi-ingredient
dietary supplement containing TCR and caffeine (150 mg) (product name = TheaTrim) to caffeine
alone and placebo, and they also found no differences in resting heart rate response across groups.
Similar results were also reported by He and colleagues in healthy adults [16]. High resting heart rate
is now considered an important constituent for increasing the chance of mortality, and this
relationship is independent of age, sex, or blood pressure in adults [35]. As DYM and TCR may
become a dietary supplement consumed on a regular basis, like caffeine is for many, establishing its
negligible impact on resting heart rate is important for maintaining health.
Resting blood pressure is also an important indicator of cardiovascular health, with chronic
elevated blood pressure (i.e., hypertension) acknowledged to increase cardiovascular disease risk.
Hypertension was recently defined as a systolic blood pressure of 130 or greater and a diastolic blood
pressure of 80 or greater [36]. Caffeine consumption is well known to acutely increase blood pressure,
likely through antagonism of the alpha-adrenergic receptors 1 and 2 (or α1 and α2) throughout the
body [37], and its ability to enhance vascular resistance raises questions regarding its impact on the
development of hypertension [37]. Therefore, the influence of TCR and DYM on blood pressure is of
interest, due to their structural similarities to caffeine. One investigation in rats examined the impact
of TCR on alpha-adrenergic receptors and found that pre-treatment caused adenosine receptor
antagonism, as demonstrated by TCR’s ability to counteract the motor depression induced by
adenosine receptor agonists (i.e., CPA and CGS-21680) [24]. Based on this evidence, one may expect
TCR, and possibly DYM, to influence systolic and diastolic blood pressure like caffeine. However,
our data do not support this hypothesis as we found no significant acute or chronic effects of DYM
alone or in combination with TCR on systolic or diastolic blood pressure. Rather, our findings appear
to corroborate those of four other studies examining TCR in humans [14,15,17,19], and one in rats
[34], which reported no observable negative effects on blood pressure [14,17], even when TCR was
combined with caffeine [15,19].
In addition to heart rate and blood pressure, a resting ECG assessment was collected prior to
supplementation and at 60 minP on each visit to further examine the acute cardiovascular safety of
DYM and its combination with TCR. Again, no abnormal changes in electrical activity of the heart
were observed for any experimental group across visits. The International Conference for
Harmonization E14 highlighted the importance of thorough QT/QTc assessments of new non-
antiarrhythmic drugs [38]. QT prolongation is a surrogate marker for delayed myocardial
Nutrients 2020, 12, 654 17 of 20
repolarization and is associated with a higher risk of cardiovascular events (i.e., torsades de pointes,
an uncommon form of polymorphic ventricular tachycardia) and mortality in not only cardiac
patients, but also the general population [39]. While both DYM and TCR are categorized as dietary
supplements, their potential stimulatory effects warranted their QTc examination. When groups were
combined, ECG QTc assessments indicated a significant average drop of ~4 ms during the 60-minP
assessment on Visit 2. This change is within the threshold level of regulatory concern often used
within thorough QT/QTc studies (i.e., 5 ms) [38]. Overall, parameters associated with electrical
conduction of the heart remained within normal parameters, despite the supplement consumed.
4.2. Blood Biomarkers
Hematological markers associated with health were examined, with no abnormal results found
following 28 days of supplementation. Mean values fell within normal clinical reference ranges for
all markers associated with CBC, CMP, and the lipid panel, excluding hemoglobin at Visit 3 for the
group consuming 150 mg of DYM and calcium for the 100-mg DYM group. Changes were not
significant, and we do not suspect DYM was responsible for these concentration fluctuations.
Furthermore, collapsed results across groups indicate increased mean corpuscular volume, mean
corpuscular hemoglobin, absolute eosinophils, creatinine, estimated glomerular filtration rate,
chloride, total carbon dioxide, bilirubin, alanine aminotransferase, and high density lipoprotein
(HDL) on Visit 3; however, no values fell outside what was deemed healthy for participants involved
in this study and would likely be considered stochastic. Researchers examining clinical hematological
data in male and female rats supplemented with DYM for 90 days (0, 75, 112, 150, 187, and 225 mg/kg)
and two additional groups of five rats per sex that were administered 0 and 225 mg/kg for further
evaluation following a 28-day recovery period found that the majority of the hematological results
remained within or marginal to historical control ranges, suggesting no toxicity concerns [25]. In the
only human study examining the effect of TCR on clinical blood markers, Taylor et al. [17] reported
no concerns related to dosages of 200 and 300 mg over eight weeks. Interestingly, individuals
consuming the 300-mg dose of TCR experienced decreases in total cholesterol and low-density
lipoproteins, at the four- and eight-week assessments, respectively. While our results did not indicate
changes in these lipid markers, collapsed results indicated an increase in high-density lipoproteins.
More research should be conducted on DYM and TCR alone and in combination as it relates to
cholesterol, as tea (e.g., green tea), which contains alkaloids, was demonstrated to beneficially
influence cholesterol profiles [40].
5. Conclusions, Limitations, and Future Directions
This is the first study to examine DYM alone or in combination with TCR in humans. Our
findings indicate that once-daily supplementation with DYM alone (100 mg and 150 mg) or in
combination with TCR (DYM 100 mg + TCR 50 mg or DYM 150 mg + 25 mg TCR) consumed by
apparently healthy, young men and women does not negatively impact health, as measured by
cardiovascular function and a comprehensive hematological panel.
As with all studies, there are limitations to our research which we openly acknowledge. Firstly,
our safety assessments of health were limited to tests of cardiovascular function (heart rate, blood
pressure, ECG) and routine blood work (complete blood count, comprehensive metabolic panel, and
lipid panel). Secondly, our subject sample included only younger men and women; future studies
may consider examining older adults, as both younger and older individuals may be interested in
using TCR and DYM. Thirdly, our participant inclusion criteria ranged from sedentary to
recreationally active individuals. As exercise may impact caffeine kinetics, including potentiation of
its action, reduction in the extent of its effects, and acceleration of its elimination, there is the
possibility that participants who engaged in more exercise than others (i.e., sedentary) experienced
different rates of metabolism and excretion of their designated supplement (due to the similar
structures to caffeine), which may have impacted the results. Finally, we recommend reproducing
our study design with larger sample sizes to more thoroughly interrogate the effects of DYM and
TCR on health. In addition, future studies should examine the impact of DYM alone or in combination
Nutrients 2020, 12, 654 18 of 20
with TCR, and possibly caffeine, on cognitive function and exercise performance, as these ingredients
are currently being included in pre-workout and nootropic supplements.
Author Contributions: All authors read and agreed to the published version of the manuscript.
Conceptualization, T.A.V., M.T.S., Y.F., T.A.E., and K.R.T.; methodology, T.A.V., M.T.S., Y.F., T.A.E., A.J.H., and
K.R.T.; formal analysis, G.T.M.; investigation, T.A.V., M.T.S., A.R.B., A.J.H., M.G.A., Y.F., M.B., K.R.T., M.S., and
A.S.M.; resources, K.R.T. and Y.F.; data curation, T.A.V., M.T.S., A.R.B., A.J.H., M.G.A., M.B., M.S., and A.S.M.;
writing—original draft preparation, T.A.V.; writing—review and editing, T.A.V., M.T.S., A.R.B., A.J.H., M.G.A.,
Y.F., G.T.M., G.M.H., T.A.E., M.B., K.R.T., M.S., and A.S.M.; supervision, T.A.V., T.A.E., G.M.H., and Y.F.; project
administration, T.A.V.; funding acquisition, T.A.V. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by Compound Solutions, Inc. (Carlsbad, CA). Compound Solutions, Inc.
also provided the supplements for this study. No member of Compound Solutions, Inc. was involved in data
collection, interpretation of results, or manuscript writing.
Acknowledgments: The authors would like to thank the participants who kindly donated their time to complete
the study.
Conflicts of Interest: T.A.V. received a grant from Compound Solutions, Inc. to complete this research. All
funding was handled by the Kennesaw State University Grant’s Office. No authors, including T.A.V, have a
financial or business interest related to the studied products.
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