Effect of the dietary supplement ElevATP on blood ATP level: An acute pilot clinical study

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Journal of Aging Research & Clinical Practice©
EFFECT OF THE DIETARY SUPPLEMENT ELEVATP ON BLOOD ATP
LEVEL: AN ACUTE PILOT CLINICAL STUDY
T. Reyes-Izquierdo1, B. Nemzer2, R. Argumedo1, C. Shu1, L. Huynh1, Z. Pietrzkowski1
Introduction
elevATP™ is a blend of plant bio-inorganic trace
minerals and polyphenol-rich apple extracts . The plant
mineral portion has previously been reported by our
research team to have potential to increase blood levels of
ATP in human subjects. Additionally, dietary
polyphenols are widely distributed in fruits, wine, tea,
vegetables and fruits and possess many biological
functions, (for review see (1)). Recently, polyphenols
have been shown to play an important role in the
functioning of mitochondria (2-6).
Mitochondria are the primary energy generating
organelles of the cell, producing ATP through a chain of
enzyme complexes. These enzymes require metals such
as iron, copper and manganese for catalytic activities.
However, mitochondria are highly sensitive to oxidative
damage and must balance the availability of transition
metals with the generation of reactive oxygen species
(ROS) (7). ATP not only is an intracellular energy carrier
and participates in hundreds of biochemical reactions (8),
but also has a number of extracellular functions. ATP is
typically released in response to various stimuli, such as
mechanical pressure, or after treatment with agonists,
such as serotonin and acetylcholine (9). Extracellular ATP
is a requirement for several physiological processes, such
as platelet aggregation, peripheral and central
neurotransmission, clot formation, cell recognition and
immune responses (10-15).
During the process of aging, intracellular ATP
decreases and the ability to generate ATP is diminished
(9, 16, 17). While this affects intracellular processes, it also
suggests that the ability to release ATP to the
extracellular milieu for regulatory processes might be
limited in aged cells and tissues. Consequently, basic and
clinical research has focused on ATP supplementation as
means to promote muscle energy metabolism and
healthy aging in humans (18).
Previous studies have described conflicting results
regarding the use of exogenous ATP as a dietary
supplement (19). It has been reported that chronic intake
of exogenous ATP can cause alterations in blood
oxygenation, peripheral blood flow and muscle
metabolism (9). Also, an increase of ATP production is
associated with an increase of intracellular ROS (20),
which can compromise the integrity of cells by inducing
oxidative stress and causing cellular dysfunction (21).
Because of the limitations of direct ATP supplementation,
some groups have turned to indirect approaches to
1. Applied BioClinical Inc., 16259 Laguna Canyon Rd, Irvine, CA, USA 92618;
2. FutureCeuticals Inc., 2692 N. State Rt. 1-17., Momence, IL, USA 60954
Corresponding Author: Tania Reyes-Izquierdo, 16259 Laguna Canyon Rd, Irvine
CA, 92618 USA, Phone (1) 949 502 4496, Fax (1) 949 502 4987,
Email: tania@abclinicaldiscovery.com
1
Abstract: Objectives: Adenosine triphosphate (ATP) participates in a number of biological processes and its levels diminish during
aging. We studied the effects of a proprietary combination of a plant-mineral-rich ancient peat material and a polyphenol-rich
apple extract, marketed under the trade name elevATP™, on blood ATP levels. Design: Acute, placebo-controlled, prospective
clinical trial. Participants: 18 generally healthy, adult human subjects. Intervention: A single, 150 mg dose of elevATP™ or 50 mg of
encapsulated silica oxide (placebo). Measurements: Blood was collected prior to, and at 60, 90 and 120 minutes after treatment. We
measured whole blood ATP, total mammalian target for rapamycin (mTOR), lactate, reactive oxygen species (ROS), and glucose.
We also identified and quantified the mineral and bioactive components of elevATP™. Results: When compared to the placebo
group, elevATP™ caused an acute increase in blood levels of ATP by 64% (P=0.02). ROS and lactate levels were unchanged by
elevATP. Total mTOR levels in blood were modestly, but significantly, lower after treatment. Conclusion: Results show that
treatment with a single dose of elevATP™ increased blood ATP levels without increasing ROS. Confirmation of these results in a
larger study sample is needed. Trials in older individuals may be particularly informative.
Key words: Blood total ATP, blood ROS, blood lactate, blood total mTOR, healthy aging, micronutrients, polyphenols.
Received November 30, 2012
Accepted for publication February 21, 2013
REYES_04 LORD_c 15/04/13 13:51 Page1

increase physiological ATP production. Recent studies
have shown that natural supplements such as
polyphenols can enhance and increase the concentration
of ATP, as well as lower the activity of lactate
dehydrogenase (LDH) and creatine pyruvic kinase (CPK)
(21). Our research team is investigating various types of
natural products capable of increasing endogenous pools
of intracellular ATP, without increasing the production of
ROS (22, 23).
In this study, 18 healthy fasting subjects were given a
single encapsulated dose of 150 mg of elevATP™ or
placebo. We report that elevATP™ significantly increased
blood ATP levels with respect to the baseline and versus
the placebo, while reducing mammalian target for
rapamycin (mTOR) levels and showing no statistically
significant effect on serum level of lactate and ROS.
Materials and Methods
Materials
elevATP™ powder was provided by FutureCeuticals,
Inc., Momence, IL USA. Dulbecco's phosphate buffered
saline (PBS), phenyl methane-sulfonyl-fluoride (PMSF),
dimethyl sulfoxide (DMSO), 200% Proof ethanol;
leupeptin and water were purchased from Sigma Chem.
Co. (St Louis, MO, USA). 5-O-Caffeoylquinic acid, Gallic
acid and quercetin-3-glucoside were purchased from
Sigma Aldrich (Poole, UK). (–)-Epicatechin and Phloretin-
2’-O-glucoside were purchased from Extrasynthese,
(Genay, France).
Methanol and acetonitrile were obtained from
Rathburn Chemicals (Walkburn, Scotland). Formic acid
was obtained from Fisher Scientific (Loughborough, UK).
Protein Low Binding microtubes were obtained from
Eppendorf (Hauppauge, NY, USA) and RC DC Protein
Assay Kit II was from Bio-Rad (Palo Alto, CA, USA).
Intracellular ROS kits were purchased from Cell Biolabs
(San Diego, CA, USA). ATP-luciferase assays were
obtained from Calbiochem (San Diego, CA, USA).
Heparin and “dry” blood collection tubes were obtained
from BD Vacutainer (Franklin Lakes, NJ, USA). Total
mTOR ELISA kits were purchased from Cell Signaling
Technology® (Danvers, MA, USA). Portable gas meter
and CG4+ cartridges were from Abbott Laboratories
(Abbott Park, IL, USA).
ElevATP™ Mineral Analysis
A 1.2 g sample test portion of ElevATP™ was dry-
ashed at 500°C ± 50°C for 8 hours and treated with nitric
acid. The resultant ash was treated with concentrated
hydrochloric acid (5%), dried, and redissolved in
hydrochloric acid solution (24). The amount of each
element was determined by comparing the emission of
the unknown sample against the emission of each
element from standard solutions using Inductively
Coupled Plasma Atomic Emission Spectroscopy (ICAP-
61E-Trace, Thermo Jarrell-Ash) (25)or by mass
spectrometry (USP <730>). All standard solutions used
were obtained from Inorganic Ventures (Christiansburg,
VA, USA) and were of analytical-reagent grade. The RSD
for analysis of each element was 4.8%
Polyphenols Analysis
Polyphenol analysis was carried out on a Thermo
Surveyor HPLC system comprising of an autosampler
with sampler cooler maintained at 6 °C and a photodiode
array detector scanning from 200-600 nm. Samples (5 or
10μl) were injected onto a 250 x 4.6mm C18 RP Polar
Column (Phenomenex; Torrance, CA, USA) maintained at
40 °C and eluted with a 5-40% gradient of 0.1% formic
acid and acetonitrile at 1 mL/min over 45 minutes. The
eluted sample passed serially through an absorbance
detector and then a fluorescence detector (Jasco, Japan;
excitation λ290 nm, emission λ320 nm). Twenty percent
of the sample was diverted to the electrospray interface of
the mass spectrometer. All samples were run in negative
ion mode using data-dependent MS-MS for compound
identification. The scan range was from 150-1500 amu.
Samples of apple extract were also analyzed using a
UHPLC system (Thermo Fisher Scientific, San Jose, CA,
USA) with an Orbitrap Exactive mass spectrometer. In
this system, a 2 mm version of the column described
above was used with the same mobile phase gradient
running at a reduced rate of 200 mcL/min. Identifications
are based on co-chromatography with authentic
standards and from comparison of exact mass or MS-MS
spectra with previously published data (26).
Quantification with authentic standards was carried out
on both absorbance and fluorescence data. Quantification
of catechins and procyanindins in the UHPLC system
was carried out in the mass spectrometer.
Clinical Study
Inclusion and Exclusion Criteria
This study was conducted according to the guidelines
laid down in the Declaration of Helsinki and all
procedures involving human subjects were approved by
the Institutional Review Board at Vita Clinical SA,
Avenida Circunvalacion Norte #135, Guadalajara, JAL,
Mexico 44270 (Study protocol ABC-NCI-12-14-ATP).
Eighteen study participants were selected. They were
generally healthy, not using any type of medication or
supplements for a period of at least 15 days prior to the
start of the study, with ages between 21 and 55 and a BMI
between 21 and 30 kg/m² (SD ±5.88). Participants were
ELEVATP ON ATP BLOOD LEVELS.
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excluded if they self-reported symptoms or carried an
active diagnosis of rhinitis, influenza, other acute
infections, or diabetes mellitus. Subjects were also
excluded if they reported allergies to dietary products.
Subjects were excluded upon the use of anti-
inflammatories, analgesics, statins, diabetic drugs, anti-
allergy medicines, multivitamins or supplements rich in
polyphenols.
Blood Collection
Enrolled participants were instructed not to eat for
12h prior to the initial blood draw. Body temperature and
blood samples were taken prior to and during treatment.
After participants gave written consent, subjects were
randomly assigned to either the treatment or placebo
group with similar characteristics for age and weight in
both groups. The placebo group took 50 mg of
encapsulated silica oxide, while the treatment group
ingested 150 mg of encapsulated elevATP™. Participants
in both groups received 200 ml water to swallow with the
test capsule.
Four hundred microliters of blood were collected by
finger puncture and placed in Safe-T-Fill® Capillary
blood collection tubes (Ram Scientific Inc. Yonkers, NY)
or 100 μL heparin-sulfate capillary tubes (Fisher
Scientific). Samples were collected at each of four time
points: immediately prior to test capsule administration
(T0) and at 60, 90 and 120 minutes. Immediately after
collection, blood samples were either snap frozen for ATP
and ROS assays or further processed for total mTOR
assays. Participants remained at rest during testing.
ATP Detection and Quantification
ATP concentration was determined using an ATP
Assay Kit (Calbiochem, San Diego, CA, USA) with a
modification to the original method, as previously
described (23). Briefly, 10 μL of lysed blood and 100 µL
ATP nucleotide-releasing buffer containing 1 µL
luciferase enzyme mix were added to a white plate and
immediately placed on a luminometer (LMaX, Molecular
Devices; Sunnyvale CA, USA). A kinetic assay was read
at 470 nm for 15 min at 3 min intervals. Relative Light
Units (RLU) were recorded and ATP concentrations
determined in comparison to an standard curve for ATP.
ROS Detection
ROS were detected by using a cell based ROS assay kit
(Cell Biolabs, San Diego, CA, USA) with modifications to
the original method, as previously described (23). Briefly,
10 μL of diluted whole blood (1:100 in water) was mixed
with 100 μL 2’, 7’-Dichlorodihydrofluorescein diacetate
(DCFH-DA) 1X in water in a clear bottom black plate
(Rochester, NY USA). This mixture was immediately
placed in a fluorescence spectrophotometer (Molecular
Devices, Sunnyvale, CA, USA) and a kinetic assay was
run, recording Excitation/Emission (Ex/Em) at 480/530
nm for 60 min at 5 min intervals. ROS concentration was
determined by comparison to a 2’, 7’-
Dichlorodihydrofluorescein (DCF) Standard Curve.
Lactate Detection
For the determination of lactate levels, finger blood
samples were analyzed with an i-STAT clinical blood gas
analyzer (Abbott Laboratories, Abbott Park, IL, USA).
100µL of blood were loaded in CG4+ Cartridges (Abbot
Laboratories, NJ, USA) and tested for lactate.
Total mTOR Detection
For Total mTOR analysis, cell lysates were prepared
according the instructions included in the kit. Briefly, 100
μL of whole blood were added to 900uL of 1X Cell Lysis
Buffer, containing 1 mM PMSF into a 1.5 ml tube.
Samples were placed in an ice bath and sonicated for 5
minutes. Afterwards, cell lysates were centrifuged at
14,000 x g for 10 minutes at 4 °C. The supernatant was
used for Total mTOR determination, according to the
manufacturer’s instructions.
Results
Chemical Analysis
We determined the mineral (Table 1) and bio-active
compound (Table 2) content of elevATP™. A total of 66
chemical elements were simultaneously assayed after
acid mineralization using both ICP-OES and ICP-MS
(Table 1). The total element content was 450,235 mg/kg,
as determined by adding the concentrations of each
element. The ICP-OES was used to determine 33
elements, while the remaining 33 elements were
determined by using ICP-MS. The total amount of six
macro-nutrient minerals (Ca, P, Na, K, Mg and S) was
424,087 mg/kg and the total amount of ten micro-
nutrient minerals (B, Co, Cr, Cu, I, Fe, Mn, Mo, Se, and
Zn) was 26,148 mg/kg.
The main plant phenolic component of elevATP™ by
weight; was chlorogenic acid (5-O-caffeoylquinic acid),
having a concentration of 201±11 mg / 100g.
Procyanidins were the second most abundant phenolics
with concentrations of dimers and trimers of 127 ± 1 mg /
100 g and 30 ± 0 mg / 100 g, respectively. The other major
phenolic compounds were hydroxycinnamic acids,
specifically chlorogenic and coumaric acids. ElevATP™
contained two catechins, (+)catechin and (-) epicatechin.
Flavonol (quercetin) and dihydrochalcones (phloretin
REYES_04 LORD_c 15/04/13 13:51 Page3

and phloridzin) were detected in trace amounts of 39±4
mg / 100 g and 18±0 mg / 100 g, respectively.
Table 1
Mineral composition of elevATP™
Mineral Concentration Mineral Concentration
(mg/kg) (mg/kg)
Aluminum 16,463 Mercury <0.01
Antimony 0.05 Molybdenum 0.07
Arsenic 0.58 Neodymium 1.37
Barium 15.44 Nickel 78.03
Beryllium 5.22 Niobium 0.91
Bismuth 2.04 Osmium 0.01
Boron 27.65 Palladium 0.05
Bromine 7.08 Phosphorus 224.41
Cadmium 2.13 Potassium 1,402
Calcium 11,831 Praseodymium 3.38
Cerium 4.76 Rhenium 0.02
Cesium 0.23 Rhodium 0.01
Chromium 19.73 Rubidium 7.79
Cobalt 38.12 Ruthenium 0.03
Copper 6.46 Samarium 2.78
Dysprosium 4.58 Scandium 1.15
Erbium 2.66 Selenium 2.56
Europium 0.86 Silicom 741.51
Gadolinium 4.66 Silver 0.12
Gallium 23.36 Sodium 36,820
Germanium 30.39 Strontium 63.64
Gold 3.73 Sulfur 249,100
Hafnium 0.96 Tantalum 0.31
Holmium 0.24 Terbium 0.29
Indium 0.11 Thorium 1.78
Iron 6,240 Thulium 0.21
Iodine 2.82 Tin 0.04
Lantharum 5.87 Tungsten 1.35
Lead <0.05 Vanadium 0.05
Lithium 235.01 Ytterbium 2.26
Lutetium 0.31 Yttrium 23.25
Magnesium 124,710 Zinc 391.72
Manganese 1,674 Zirconium 1.67
Table 2
Bioactive compounds in elevATP™
Analyte Concentration in mg/100 g
5-O-caffeoylquinic acid 201 ± 11
Procyanidin dimers 127 ± 1
Procyanidin trimers 30 ± 0
Catechin 27 ± 2
Epicatechin 50 ± 1
4-O-p Coumaric acid 25 ± 2
Phloretin xyloglucoside 8 ± 0
Phloretin glucoside 8 ± 0
Quercetin 39 ± 4
Phloretin 2 ± 0
Effect of elevATP™ in Humans
Eighteen subjects were included in this clinical study.
Participants (10 male and 8 female) with ages >19 and
<59 had a mean BMI of 26.43 (SD 5.88). Participants were
randomly assigned, four female and five male per group
to two groups. The placebo group (n = 9) received 50 mg
of silica oxide and the test group (n= 9) received 150 mg
of elevATP™. ATP levels obtained from samples
collected at 60, 90, and 120 minutes were averaged and
compared the effect of elevATP™ to placebo. Blood ATP
levels in the elevATP™ treatment group increased by
64% (Figure 1), which was statistically significant
(p=0.016) when compared to the placebo. Blood ATP
levels did not increase significantly in the placebo group.
Since higher levels of ATP have been associated with
an increase in free radicals, we measured ROS in blood.
Samples were normalized as the percent change over
baseline at time zero. The elevATP™ treated group had
10% lower ROS levels than the silica-treated group, a
difference that was statistically significant (Student’s t-
test; p=0.011). It is important to note that ROS levels were
105% of baseline in the placebo group and 95% of
baseline in the treatment group, both of which reflected
insignificant changes from baseline (Figure 2).
Figure 1
Effect of elevATP™ on blood ATP levels. Whole blood
was collected from placebo-treated or elevATP™-treated
subjects at T0 (baseline), T60, T90 and T120. ATP was
detected by using a luciferase-based assay on 10μl of
lysed whole blood. Data from T60, T90 and T120 were
compared to baseline and pooled for comparison
between treatment groups. ATP was significantly higher
after treatment with elevATP™ (p=0.016). Data are
presented as Mean +/- SE, n=9
Lactate levels were 11% higher than baseline in the
treatment group, while levels in the placebo group were
9% lower (Figure 3). The differences between groups
were not statistically significant (p=0.081). The
mammalian target of rapamycin (mTOR) was also
measured, since it can act as an ATP sensor. As shown in
Figure 4, total mTOR remained unchanged in the both
groups compared to baseline. The placebo group had a
non-significant increase of 5% compared to the baseline
and the elevATP™ group showed a slight decrease of 3%.
However, when compared with each other, differences
were statistically significant (p=0.021). When compared
with placebo group, the treated group showed no
statistically significant difference in blood glucose levels
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(p=0.898). In both groups, glucose levels remained stable,
as can be observed in Figure 5.
Figure 2
Effect of elevATP™ on concentration of ROS in whole
bloo. Reactive oxygen species (ROS) were also detected
after treatment with placebo or elevATP™. The placebo
group showed a slight increase in ROS (5% over baseline)
and the elevATP™ group showed a decrease (5% below
baseline). Although a statistical significance was observed
(p=0.011) when comparing the placebo to the elevATP™
group, when comparing these differences against the
baseline, they are quite insignificant. Data are presented
as Mean +/- SE, n=9
Figure 3
Plasma lactate levels after treatment with elevATP™.
Lactate levels were also detected in plasma after
treatment with elevATP™ and placebo. Although lactate
levels were higher than baseline in the elevATP™ group
by 11%, while levels in the placebo group were lower by
9%, the differences between groups were not statistically
significant (p=0.081). Data are presented as Mean +/-
SE,n=9
Discussion
Mitochondria are the primary location for the
production of ATP molecules in most cells, carried out by
enzymatic reactions. Although these enzymes require
transition metals such as iron, copper and manganese for
their performance, they are highly sensitive to oxidative
damage (7). Recently, polyphenols have been described
as important role-playing molecules in the functioning of
mitochondria (3, 27, 28) possessing excellent antioxidant
potency (21). Selected targets included blood ATP, ROS,
lactate, mTOR and glucose. Most of the ATP in blood is
confined to the red blood cells (17, 29, 30, 31, #82, 32, 33).
However, extracellular concentration of ATP has been
also detected (34-38).
Figure 4
Total mTOR after treatment with elevATP™. Total
mammalian target of rapamycin (Total mTOR) remained
unaffected in both groups compared to baseline. The
placebo showed a non-significant increase of 5%
compared to the baseline and the elevATP™ group
showed a slight decrease of 3%. However, when
compared with each other, the decrease in mTOR in the
elevATP™ groupwas statistically significant (p=0.021).
Data are presented as Mean +/- SE, n=9
Figure 5
Glucose after treatment with elevATP™. Blood glucose
was monitored over the duration of the study. In both
groups, glucose levels remained stable and the
differences between the placebo and elevATP™ treated
groups were not significant (p=0.898). Data are presented
as Mean +/- SE, n=9
In this study, total ATP was measured in whole blood
immediately after collection, as previously described (22).
Blood collected from subjects treated with elevATP™
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showed an increase in blood ATP (up to 45% compared to
baseline). However, this was not observed in the group
treated with placebo (Fig 1). This result suggests that
elevATP™ could positively affect the process of ATP
generation in whole blood, assuming that the chemical
components present in elevATP™ are delivered quickly
to blood stream. Hypothetically, elevATP™ could be
used for stimulation of ATP in blood cells and possibly in
other tissues and organs. However, a larger study is
required to confirm the preliminary results of this clinical
pilot study, which could identify possible mechanisms of
action and verify whether ATP is increased in other
tissues, such as skin or adipose tissues.
Studies of ATP levels in various states such as cancer
(29, 39-41), systemic lupus (42), diabetes type II (17), and
exercise performance (9, 21 , 43) broadly suggest that
increased ATP levels correlate with health and
performance. Likewise, ATP-producing ability of organs
and tissues diminishes considerably with age (32).
Growing evidence suggests that endogenous oxidants,
such as hydroxyl radicals and hydrogen peroxide (HO-),
superoxide (O2-) and singlet oxygen (1O2), accelerate the
aging process by damaging cell macromolecules such as
proteins, DNA and lipids (16). As the main source of ATP
production switches from carbohydrate sources to fatty
acids, the amount of free radicals generated increases (16)
in all tissues, including blood (44). Moreover, the high
oxygen tension in blood and iron in heme is a net
oxidative environment, from both non-enzymatic and
enzymatic pathways, despite lacking mitochondria.
Mitochondria are the main source of oxidants in most
non-blood cells and their integrity declines with age.
With a loss of mitochondrial integrity, ATP synthesis is
impaired while reactive oxygen species levels increase
(45).
In our study, ROS were lower in the elevATP™ treated
group compared to their baseline level. ROS levels in the
placebo group were unchanged. These results suggest
that the increase of ATP observed (Fig 1) does not result
in a concomitant increase in ROS. This result has two
possible explanations. It could be that elevATP™
increases blood ATP levels in a manner that is unrelated
to ROS production. On the other hand, elevATP™
contains a number of potent compounds which could be
directly scavenging and reducing ROS, thereby lowering
their levels overall. Indeed both processes could be taking
place. In any case, previous studies with ATP
supplementation have resulted in undesirable increases
in ROS. Therefore, elevATP™ may be a good candidate to
enhance blood ATP levels without causing a concomitant
increase in ROS.
mTOR is a member of the phosphoinositide kinase-
related kinase (PIKK) family that functions as a central
element in a signaling pathway involved in the control of
many processes, including protein synthesis and
autophagy (46). It has been described as a sensor of
energy levels in the cell (47) and its activity increases in
diseases such as cancer and diabetes. In certain cellular
and animals systems, it also correlates with the speed of
aging (48). mTOR activity can be affected by dietary
microelements (49, 50). It has also been reported that
dietary polyphenols and microelements can promote
healthy levels of mTOR and enhance protein synthesis
(51, 52). Our results show that elevATP™ reduced total
mTOR in blood, an effect not seen in the placebo group
(Fig 4). While this reduction was modest, it was
statistically significant. These results suggest that the use
of elevATP™ as a nutritional supplement may result in
reducing mTOR levels. These results suggest that , a
single treatment of elevATP™ at a serving of 150 mg
resulted in a significant increase of blood level of total
ATP; with no concomitant increase in blood ROS or
serum lactate and a reduction of total mTOR levels in
blood under these experimental conditions. These results
should be considered preliminary and should be
confirmed in larger clinical testing.
Acknowledgements: This study was funded by FutureCeuticals, Inc. We would
like to thank Michael Sapko for his help in editing the manuscript. All authors
declare that they have no conflicts of interest.
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