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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©
T. Reyes-Izquierdo1, B. Nemzer2, R. Argumedo1, C. Shu1, L. Huynh1, Z. Pietrzkowski1
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,
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
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
REYES_04 LORD_c 15/04/13 13:51 Page2
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
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.
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
REYES_04 LORD_c 15/04/13 13:51 Page4
(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 +/-
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™
REYES_04 LORD_c 15/04/13 13:51 Page5
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
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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... The ability to generate ATP is diminished as well, negatively impacting skeletal muscle work output capabilities [2,3]. Maintenance of higher level of ATP production in vivo is thought to correlate with better overall health and athletic performance [4]. ...
... ElevATP ™ is a blend of plant bio-inorganic trace minerals and a polyphenol-rich apple extract [4]. A single oral dose of ElevATP ™ has been shown to increase whole blood ATP levels as well as muscle levels of ATP in healthy resting subjects [4,9]. ...
... ElevATP ™ is a blend of plant bio-inorganic trace minerals and a polyphenol-rich apple extract [4]. A single oral dose of ElevATP ™ has been shown to increase whole blood ATP levels as well as muscle levels of ATP in healthy resting subjects [4,9]. It was also reported that once daily dosing with ElevATP ™ for twelve weeks resulted in improved resistance training-induced skeletal muscle hypertrophy without affecting fat mass or blood chemistry [9]. ...
Full-text available
Background: Adenosine 5’-triphosphate (ATP) serves as a cellular source of energy for metabolic processes. Maintaining high levels of intracellular ATP has the potential to enhance performance. ElevATP™, a proprietary blend of ancient peat and apple extract, has twice been reported to increase intracellular blood levels of ATP in resting subjects after a single dose per os. More recently, resistance-trained males supplemented with ElevATP™ for 12 weeks, have shown increased resistance training-induced skeletal muscle hypertrophy. For this pilot study, we determined the acute effects of a single dose of ElevATP™ on the exercise output of subjects with sedentary lifestyles. Methods and findings: Nine healthy subjects were evaluated before, during, and after 20 minute duration stepping exercise in a blinded three-way crossover study of placebo and a single dose of ElevATP™. Two lots of ElevATP™ (Lot A and Lot B) were each compared to placebo in all subjects. Four (4) female and five (5) male subjects that did not regularly exercise were evaluated. Mean subject age was 27.3 ± 5.0 years and BMI was 28.97 ± 6.6 kg/m². ElevATP™ supplemented subjects (Lot A and Lot B) took a significantly greater number of steps and burned more calories than when treated with placebo. Post-exercise analysis has shown no significant changes in blood lactate or glucose levels between ElevATP™ (Lota A or Lot B) and placebo treatments. Conclusion: All nine subjects experienced an increase in exercise performance after ingesting a single dose of either of the two tested lots of ElevATP™, compared to a placebo control.
... Therefore, supplementation strategies for increasing endogenous ATP levels may be desirable. Previously, oral supplementation with a proprietary blend of ancient peat and apple extracts have been demonstrated to increase intracellular ATP levels in whole blood and intramuscular levels of ATP in resting subjects, suggesting increased activity of bodily processes that lead to endogenous ATP production [8,9]. ...
... Preliminary reports from this laboratory suggest this occurs without an increase in reactive oxygen species, which may be associated with increased ATP production [10]. In fact, ancient peat and apple extracts may actually decrease reactive oxygen species [8], possibly blunting a potential increase caused by resistance training [11]. ...
... Despite these observations, supplementation for indirect ATP enhancement is yet to be evaluated for potential to augment performance in response to resistance training. However, the existing data on ancient peat and apple extracts for increasing both whole blood and muscle ATP levels [8,9] and muscle mass [16] support the plausibility for chronic supplementation yielding positive augmentation of performance following resistance training. Therefore, the purpose of this study is to determine the effects of a proprietary blend of ancient peat and apple extracts on athletic performance. ...
Full-text available
Background Increased cellular ATP levels have the potential to enhance athletic performance. A proprietary blend of ancient peat and apple extracts has been supposed to increase ATP production. Therefore, the purpose of this investigation was to determine the effects of this supplement on athletic performance when used during 12 weeks of supervised, periodized resistance training. Methods Twenty-five healthy, resistance-trained, male subjects completed this study. Subjects supplemented once daily with either 1 serving (150 mg) of a proprietary blend of ancient peat and apple extract (TRT) or an equal-volume, visually-identical placebo (PLA) daily. Supervised resistance training consisted of 8 weeks of daily undulating periodized training followed by a 2 week overreach and a 2 week taper phase. Strength was determined using 1-repetition-maximum (1RM) testing in the barbell back squat, bench press (BP), and deadlift exercises. Peak power and peak velocity were determined during BP at 30 % 1RM and vertical jump tests as well as a 30s Wingate test, which also provided relative power (watt:mass) ResultsA group x time interaction was present for squat 1RM, deadlift 1RM, and vertical jump peak power and peak velocity. Squat and deadlift 1RM increased in TRT versus PLA from pre to post. Vertical jump peak velocity increased in TRT versus PLA from pre to week 10 as did vertical jump peak power, which also increased from pre to post. Wingate peak power and watt:mass tended to favor TRT. Conclusions Supplementing with ancient peat and apple extract while participating in periodized resistance training may enhance performance adaptations. Trial registration ID: NCT02819219, retrospectively registered on 6/29/2016
... Therefore, an indirect approach for increasing ATP levels may be desirable. Previously, Reyes-Izquierdo and colleagues determined that a 150 mg dose of a blend of ancient peat and apple extracts significantly increased blood ATP compared to placebo in 18 [31] and 20 [32] subjects. In the latter research, blood ATP increased by 40 % at 60 min following ingestion, which dropped to 28 % at 120 min following ingestion. ...
... Preliminary reports from this laboratory also support an increase in blood ATP levels, and suggest this occurs without an increase in reactive oxygen species, which may be associated with increased ATP production [12]. In fact, ancient peat and apple extracts may actually decrease reactive oxygen species [31], possibly blunting the increase caused by resistance training [3]. ...
... They were instructed to consume 1 serving (2 mL) of either PLA or TRT (150 mg ElevATP™, FutureCeuticals Inc., Momence, IL; 180 mg blend of caffeine anhydrous and PurEnergy™, Chromadex Inc., Irvine, CA; and 38 mg B vitamins) 45 min prior to training on training days or at a similar time of day on rest days. For a detailed composition of ElevATP™, composed of ancient peat and apple extracts, see [31]. The PurEnergy™ ingredient is composed of 43 % caffeine and 57 % pterostilbene. ...
Full-text available
Background Increased ATP levels may enhance training-induced muscle accretion and fat loss, and caffeine is a known ergogenic aid. A novel supplement containing ancient peat and apple extracts has reported enhanced mitochondrial ATP production and it has been coupled with an extended-release caffeine. Therefore, the purpose of this investigation was to determine the effects of this supplement on body composition when used in conjunction with 12 weeks of resistance training. Methods Twenty-one resistance-trained subjects (27.2 ± 5.6y; 173.5 ± 5.7 cm; 82.8 ± 12.0 kg) completed this study. Subjects supplemented daily with either 1 serving of the supplement (TRT), which consisted of 150 mg ancient peat and apple extracts, 180 mg blend of caffeine anhydrous and pterostilbene-bound caffeine, and 38 mg B vitamins, or an equal-volume, visually-identical placebo (PLA) 45 min prior to training or at the same time of day on rest days. Supervised resistance training consisted of 8 weeks of daily undulating periodized training followed by a 2-week overreach and a 2-week taper phase. Body composition was assessed using DEXA and ultrasound at weeks 0, 4, 8, 10, and 12. Vital signs and blood markers were assessed at weeks 0, 8, and 12. Results Significant group x time (p < 0.05) interactions were present for cross-sectional area of the rectus femoris, which increased in TRT (+1.07 cm²) versus PLA (−0.08 cm²), as well as muscle thickness (TRT: +0.49 cm; PLA: +0.04 cm). A significant group x time (p < 0.05) interaction existed for creatinine (TRT: +0.00 mg/dL; PLA: +0.15 mg/dL) and estimated glomerular filtration rate (TRT: −0.70 mL/min/1.73; PLA: −14.6 mL/min/1.73), which remained within clinical ranges, but no other significant observations were observed. Conclusions Supplementation with a combination of extended-release caffeine and ancient peat and apple extracts may enhance resistance training-induced skeletal muscle hypertrophy without adversely affecting blood chemistry.
... Therefore, an indirect approach for increasing endogenous ATP levels may be desirable. Previously, oral supplementation with a proprietary blend of ancient peat and apple extracts has been demonstrated to increase intracellular ATP levels in whole blood and muscle, suggesting increased activity of bodily processes that produce or release endogenous ATP (Reyes-Izquierdo et al. 2013. Reyes-Izquierdo et al. (2014) determined that a 150-mg dose of this blend of ancient peat and apple extracts significantly increased ATP in cellular fraction of blood compared with placebo. ...
... Preliminary reports from this laboratory suggest this occurs without increases in reactive oxygen species, which may be associated with increased ATP production (Chang et al. 2010). In fact, ancient peat and apple extracts may actually decrease reactive oxygen species (Reyes-Izquierdo et al. 2013), possibly blunting the increase in reactive oxygen species caused by resistance training (Alessio et al. 2000). ...
... Despite these observations, supplementation for indirect ATP enhancement is yet to be evaluated for potential to induce body composition changes in response to resistance training. However, the existing data on a blend of ancient peat and apple extracts for increasing both whole blood and muscle ATP levels (Reyes-Izquierdo et al. 2013) support the plausibility that chronic supplementation may yield changes in body composition. Additionally, because this is a novel ingredient, chronic supplementation must be verified as safe. ...
Full-text available
Adenosine-5'-triphosphate (ATP) is primarily known as a cellular source of energy. Increased ATP levels may have the potential to enhance body composition. A novel, proprietary blend of ancient peat and apple extracts has been reported to increase ATP levels, potentially by enhancing mitochondrial ATP production. Therefore, the purpose of this investigation was to determine the supplement's effects on body composition when consumed during 12 weeks of resistance training. Twenty-five healthy, resistance-trained, male subjects (age, 27.7 ± 4.8 years; height, 176.0 ± 6.5 cm; body mass, 83.2 ± 12.1 kg) completed this study. Subjects supplemented once daily with either 1 serving (150 mg) of a proprietary blend of ancient peat and apple extracts (TRT) or placebo (PLA). Supervised resistance training consisted of 8 weeks of daily undulating periodized training followed by a 2-week overreach and a 2-week taper phase. Body composition was assessed using dual-energy X-ray absorptiometry and ultrasound at weeks 0, 4, 8, 10, and 12. Vital signs and blood markers were assessed at weeks 0, 8, and 12. Significant group × time (p < 0.05) interactions were present for ultrasound-determined cross-sectional area, which increased in TRT (+0.91 cm(2)) versus PLA (-0.08 cm(2)), as well as muscle thickness (TRT: +0.46; PLA: +0.04 cm). A significant group × time (p < 0.05) interaction existed for creatinine (TRT: +0.06; PLA: +0.15 mg/dL), triglycerides (TRT: +24.1; PLA: -20.2 mg/dL), and very-low-density lipoprotein (TRT: +4.9; PLA: -3.9 mg/dL), which remained within clinical ranges. Supplementation with a proprietary blend of ancient peat and apple extracts may enhance resistance training-induced skeletal muscle hypertrophy without affecting fat mass or blood chemistry in healthy males.
... Although these studies' findings are compelling, none have evaluated ancient peat and apple extracts' effects on high-intensity endurance exercise in recreational or trained endurance athletes. In addition, many of the authors associated with the studies described above [26][27][28][29] acknowledged an affiliation with the manufacturer of elevATP™; therefore, more randomized, PL-controlled trials conducted in independent laboratories are necessary to confirm its efficacy for enhancing endurance performance. ...
Full-text available
This investigation aimed to determine the effect of a multi-ingredient pre-workout supplement (MIPS) on heart rate (HR), perceived exertion (RPE), lactate concentration, and time to fatigue (TTF) during a running task to volitional exhaustion. Eleven NCAA Division I cross-country runners (20 ± 2 year; height: 171 ± 14 cm; weight: 63.5 ± 9.1 kg) participated in this randomized, double-blind, placebo-controlled cross-over study. Bayesian statistical methods were utilized, and parameter estimates were interpreted as statistically significant if the 95% highest-density intervals (HDIs) did not include zero. TTF was increased in the MIPS condition with a posterior Meandiff = 154 ± 4.2 s (95% HDI: −167, 465) and a 0.84 posterior probability that the supplement would increase TTF relative to PL. Blood lactate concentration immediately post-exercise was also higher in the MIPS condition compared to PL with an estimated posterior Meandiff = 3.99 ± 2.1 mmol (95% HDI: −0.16, 7.68). There were no differences in HR or RPE between trials. These findings suggest that a MIPS ingested prior to sustained running at lactate threshold has an 84% chance of increasing TTF in highly trained runners and may allow athletes to handle a higher level of circulating lactate before reaching exhaustion.
... A recent supplement on the market is ancient peat and apple extract, which is sold under the label of "elevATP". This supplement has been shown to increase blood levels of ATP [22], increase exercise levels in sedentary individuals from a single dose [23], and enhance responses to chronic resistance training [24]. The supplement Huperzine has been shown to have positive effects on brain function, specifically decreasing cognitive deficits and oxidative stress in the rodent model after hypobaric hypoxia [25], and has been shown in conjunction with other supplements to enhance upper body strength endurance performance [6] and anaerobic sprint performance [26]. ...
Full-text available
Recently, the use of pre-workout supplements has become popular. Research has shown their ability to increase performance for single bouts but little exists showing their ability to maintain this increase in performance over multiple bouts. Purpose: To investigate the effects of supplements on power production and the maintenance of upper and lower body tasks. Methods: Twenty-three males (22.9 ± 3.6 years, 175.6 ± 6.5 cm, 86.9 ± 15.1 kg, 19.1 ± 8.4 BF% mean ± standard deviation (SD)) were familiarized with the testing protocols and maximal bench press performances were attained (109.1 ± 34.0 kg). Utilizing a double-blind crossover design, subjects completed three trials of five countermovement vertical jumps before and after a high-intensity cycle sprint protocol, which consisted of ten maximal 5 s cycle ergometer sprints utilizing 7.5% of the subject’s body weight as resistance, with 55 s of recovery between each sprint. Subjects ingested in a randomized order a commercially available pre-workout supplement (SUP), placebo + 300 mg caffeine (CAF), or a placebo (PLA). Peak power (PP), mean power (MP), and minimum power (MNP) were recorded for each sprint. Subjects performed a velocity bench press test utilizing 80% of their predetermined one repetition maximum (1RM) for 10 sets of 3 repetitions for maximal speed, with one-minute rests between sets. Maximal velocity from each set was recorded. For analysis, bike sprint and bench press data were normalized to the placebo trial. Results: Cycle sprint testing showed no significant differences through the testing sessions. In the bench press, the peak velocity was higher with both the SUP and CAF treatments compared to the placebo group (1.09 ± 0.17 SUP, 1.10 ± 0.16 CAF, and 1 ± 0 PLA, p < 0.05) and the supplement group was higher than the PLA for mean velocity (1.11 ± 0.18 SUP and 1 ± 0 PLA, p < 0.05). Vertical jump performance and lactate levels were not significantly different (RMANOVA showed no significant differences from any treatments). Conclusions: Supplementation with a pre-workout supplement or placebo with caffeine showed positive benefits in performance in bench press velocity.
... Spectra™, a "full-spectrum" antioxidant product comprised of fruit, vegetable and herb extracts, may also act in a synergistic fashion with other ingredients by reducing NO scavenging by reactive oxygen species and improving NO bioavailability [16]. Finally, ancient peat and apple fruit extract has been shown to enhance circulating ATP levels [17,18] which may augment blood flow in a NOS independent manner [19,20]. Indeed, although many PWS marketed to increase the blood flow response to resistance training focus on NO modulation, the contribution of NO to resistance exercise induced hyperemia is not absolute and likely partially includes ATP-sensitive potassium channels [21] which may interact with ingredient(s) found in a PWS (e.g., elevATP®). ...
Full-text available
Background: We sought to determine if a pre-workout supplement (PWS), containing multiple ingredients thought to enhance blood flow, increases hyperemia associated with resistance training compared to placebo (PBO). Given the potential interaction with training loads/time-under-tension, we evaluated the hyperemic response at two different loads to failure. Methods: Thirty males participated in this double-blinded study. At visit 1, participants were randomly assigned to consume PWS (Reckless™) or PBO (maltodextrin and glycine) and performed four sets of leg extensions to failure at 30% or 80% of their 1-RM 45-min thereafter. 1-wk. later (visit 2), participants consumed the same supplement as before, but exercised at the alternate load. Heart rate (HR), blood pressure (BP), femoral artery blood flow, and plasma nitrate/nitrite (NOx) were assessed at baseline (BL), 45-min post-PWS/PBO consumption (PRE), and 5-min following the last set of leg extensions (POST).Vastus lateralisnear infrared spectroscopy (NIRS) was employed during leg extension exercise. Repeated measures ANOVAs were performed with time, supplement, and load as independent variables and Bonferroni correction applied for multiplepost-hoccomparisons. Data are reported as mean ± SD. Results: With the 30% training load compared to 80%, significantly more repetitions were performed (p < 0.05), but there was no difference in total volume load (p > 0.05). NIRS derived minimum oxygenated hemoglobin (O2Hb) was lower in the 80% load condition compared to 30% for all rest intervals between sets of exercise (p < 0.0167). HR and BP did not vary as a function of supplement or load. Femoral artery blood flow at POST was higher independent of exercise load and treatment. However, a time*supplement*load interaction was observed revealing greater femoral artery blood flow with PWS compared to PBO at POST in the 80% (+56.8%;p = 0.006) but not 30% load condition (+12.7%;p = 0.476). Plasma NOx was ~3-fold higher with PWS compared to PBO at PRE and POST (p < 0.001). Conclusions: Compared to PBO, the PWS consumed herein augmented hyperemia following multiple sets to failure at 80% of 1-RM, but not 30%. This specificity may be a product of interaction with local perturbations (e.g., reduced tissue oxygenation levels [minimum O2Hb] in the 80% load condition) and/or muscle fiber recruitment.
... Silica oxide is considered an inert ingredient and is used as a binder/carrier in many pharmaceutical preparations. To our knowledge, the effect of silica on blood lactate has not been previously reported, though we have seen this phenomenon in our previous investigations (Reyes-Izquierdo 2013). This is the first report describing that a broccoli-based material may acutely increase blood levels of intracellular ATP in human subjects. ...
Full-text available
During the last decade, broccoli (Brassica oleracea L.) has been recognized as a functional food that potentially confers numerous health benefits. Broccoli contains a glucosinolate, glucoraphanin (4-methylsulfinylbutyl glucosinolate) as well as other glucosinolates, with health-promoting effects. Bioavailability of glucosinolates requires at least three hours to be identified in blood upon ingestion. In this study we tested effect of broccoli-based material on blood level of adenosine triphosphate (ATP) during first two hours upon ingestion. Twenty-two adult healthy subjects were recruited for this study. Subjects were randomly assigned to either Placebo or broccoli sprout extract (BSE) groups (n=11). Blood intracellular ATP levels, glucose, lactate and reactive oxygen species (ROS) were determined throughout the duration of the study. A single dose of 50 mg BSE increased blood levels of ATP up to 55% (p>0.0001) during the duration of the trial. This effect was not associated with any increases in blood ROS or lactate. These results indicate that a single dose of BSE may acutely increase ATP levels in circulating human blood cells without affecting ROS or lactate levels. It is hypothesized that this effect is due to the phytochemicals present in BSE that are other than glucosinolates. Further investigations are justified to identify their phytochemicals.
... ElevATP TM is a proprietary blend of plant bio-inorganic trace minerals and polyphenols, which is purported to improve mitochondrial ATP production when consumed. To our knowledge, only one study has been published on the acute effects of elevATP ingestion on blood ATP concentration in humans (24). Their results suggested that whole blood ATP levels increased by 45% after ingestion of 150 mg of elevATP. ...
Effects Of Acute Caffeine And B-vitamin Consumption On Golf Performance During A 36-hole Competitive Tournament: 3316 Board #77 May 30, 9
Gonzalez, AM, Pinzone, AG, Bram, J, Salisbury, JL, Lee, S, and Mangine, GT. Effect of multi-ingredient preworkout supplementation on repeated sprint performance in recreationally active men and women. J Strength Cond Res XX(X): 000-000, 2019-The purpose of this investigation was to examine the effects of acute supplementation of a multi-ingredient preworkout supplement (MIPS), containing a proprietary blend of ancient peat and apple extracts, creatine monohydrate, taurine, ribose, and magnesium, on sprint cycling performance. Seventeen recreationally active men and women (23.2 ± 5.9 years; 172.9 ± 14.3 cm; 82.4 ± 14.5 kg) underwent 2 testing sessions administered in a randomized, counterbalanced, double-blind fashion. Subjects were provided either MIPS or placebo (PL) one hour before performing a sprint cycling protocol, which consisted of ten 5-second "all-out" sprints interspersed by 55 seconds of unloaded pedaling. Average power (PAVG), peak power (PPK), average velocity (VAVG), and distance covered were recorded for each sprint. Separate linear mixed models revealed decrements (p < 0.05) compared to the first sprint in PAVG (75-229 W) and PPK (79-209 W) throughout all consecutive sprints after the initial sprint during PL. Likewise, diminished (p ≤ 0.029) VAVG (3.37-6.36 m·s) and distance covered (7.77-9.00 m) were noted after the third and fifth sprints, respectively, during PL. By contrast, during MIPS, only VAVG decreased (2.34-5.87 m·s, p ≤ 0.002) on consecutive sprints after the first sprint, whereas PAVG and PPK were maintained. In addition, a significant decrease (p = 0.045) in distance covered was only observed on the ninth sprint during MIPS. These data suggest that recreational athletes who consumed the MIPS formulation, one hour before a repeated sprinting session on a cycle ergometer, better maintained performance compared with PL.
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Human peripheral blood mononuclear cells (PBMC) were used as a biological model to measure intracellular ATP (iATP) reactive oxygen species (ROS), Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR), in response to acute ex vivo treatments with HH2o. "HH2o", a fermented, food-based material commercially marketed as "Mitochroma™ has been identified as a potential modulator of iATP, OCR and ROS in these cells. PBMC were exposed to HH2O (0.1, 0.01 and 0.001% w/v) and Carbonyl-cyanide p-trifluoro-methoxyphenyl-hydrazone (FCCP) or Oligomycin, two known modulators of mitochondrial activity were used as controls. Results showed that treatment of the cells with HH2O resulted in increase of iA TP up to 220% and reduction of iADP by up to 40% while iROS levels remained unchanged. At the same time, OCR level was increased up to 24% compared to initial baseline. These results suggest that HH2O acts similarly to uncoupling protein 2 (UCP2) when induced and that it may have potential use in developing health-promoting products. These results support the use of freshly isolated human PBMCs as an experimental ex vivo model to detect the effects of nutraceutical products on dynamic cell metabolites, such as oxygen consumption, ATP, ADP and ROS.
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Acute clinical testing was performed on healthy human subjects to verify whether a single 150mg dose of a proprietary formula marketed under the trade name "Mitochroma TM " (MCRC) can actually increase blood levels of ATP. Sera and blood were collected immediately prior to treatment and at times 30, 60, and 90 minutes after treatment to measure amounts of blood ATP, lactate, ROS, and pO 2 . Additionally, whole blood was collected at 270 minutes after treatment to measure expression of selected cytokines and chemokines. In comparison to the placebo group, samples collected from subjects treated with MCRC showed increased levels of total blood ATP by 12.5% on average and reduced levels of lactate up to 13%. Blood levels of ROS and pO 2 were found unchanged under these experimental conditions. Analyses of blood collected at 270 minutes showed reduced levels of MCP-1 by up to 21%, and increased levels of Interferon-alpha up to 16%. In summary, collected data shows that treatment with a single dose of MCRC resulted in an acute increase in blood levels of total blood ATP. These results justify further clinical studies on MCRC in order to determine the effects on a more narrowly selected subject population with reduced blood levels of ATP and increased blood levels of MCP-1.
mTOR signalling is implicated in the development of disease and in lifespan extension in model organisms. This pathway has been associated with human diseases such as diabetes and cancer, but has not been investigated for its impact on longevity per se. Here, we investigated whether transcriptional variation within the mTOR pathway is associated with human longevity using whole blood samples from the Leiden Longevity Study (LLS). This is an unique cohort of Dutch families with extended survival across generations, decreased morbidity and beneficial metabolic profiles in middle-age. By comparing mRNA levels of nonagenarians and middle-aged controls, the mTOR signalling gene set was found to associate with old age (p=4.6 x 10(-7) ). Single gene analysis showed that seven out of 40 mTOR pathway genes had a significant differential expression of at least 5%. Of these, the RPTOR (Raptor) gene was found to be differentially expressed also when the offspring of nonagenarians was compared to their spouses, indicating association with familial longevity in middle-age. This association was not explained by variation between the groups in the prevalence of type 2 diabetes and cancer or glucose levels. Thus, the mTOR pathway not only plays a role in the regulation of disease and aging in animal models, but also in human health and longevity. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
We have developed an integrated package that includes software programs and algorithms for measuring in vivo total blood ATP, extracellular blood ATP, blood ATP release rates, and plasma ATP breakdown rates. This clinically tested methodology improves upon existing luciferin/luciferase and high-performance liquid chromatography assay techniques in that it involves the multidetector luminometric system for determination of blood ATP parameters with simultaneous monitoring of ATP standards and standard curve generations with r2 > 0.99 and increased speed of preparation and assay time (time necessarily for 96-well plate preparation, assay itself, and analysis). The system and associated software program permits analysis blood ATP parameters in mol, mol/L, or mol/red blood cell (RBC) and has been extensively tested with samples measured in clinics and with samples obtained and measured at the patient bedside. The mobility of the system and ability to perform measurements on location is an important consideration and feature because of the fact that ATP blood levels are rapidly hydrolyzed and hence extremely sensitive to any perturbations associated with any vigorous manipulations during the processing and analysis. EDTA inhibition of extracellular ATP hydrolysis permits determination of extracellular ATP (plasma) and ATP release rates levels performed by iterative measurements performed on sequentially diluted blood aliquots. Back-extrapolation permits close approximation of the in vivo blood ATP parameters. The assay system is capable of detecting RBC ATP release rates as low as 10,000 ATP molecules·RBC−1min−1. Drug Dev. Res. 59:152–160, 2003. © 2003 Wiley-Liss, Inc.
Objective Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (ΔΨm) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE.Methods We assessed the ΔΨm with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin-luciferase assay.ResultsBoth ΔΨm and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased ΔΨm and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and ΔΨm elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the ΔΨm elevation in lupus. CD3:CD28 costimulation led to transient elevation of the ΔΨm, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1–ATPase, had similar effects.ConclusionT cell activation and apoptosis are mediated by ΔΨm elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE.
Transition metals are essential to many biological processes in almost all organisms from bacteria to humans. Their versatility, which arises from an ability to undergo reduction-oxidation chemistry, enables them to act as critical cofactors of enzymes throughout the cell. Accumulation of metals, however, can also lead to oxidative stress and cellular damage. The importance of metals to both enzymatic reactions and oxidative stress makes them key players in mitochondria. Mitochondria are the primary energy-generating organelles of the cell that produce ATP through a chain of enzymatic complexes that require transition metals, and are highly sensitive to oxidative damage. Moreover, the heart is one of the most mitochondrially-rich tissues in the body, making metals of particular importance to cardiac function. In this review, we focus on the current knowledge about the role of transition metals (specifically iron, copper, and manganese) in mitochondrial metabolism in the heart. This article is part of a Special Issue entitled 'Focus on Cardiac Metabolism'.
Autophagy is a lysosomal degradation process that evolved as a starvation response in lower eukaryotes and has gained numerous functions in higher organisms. In animals, autophagy works as a central process in cellular quality control by removing waste or excess proteins and organelles. Impaired autophagy and the age-related decline of this pathway favour the pathogenesis of many diseases that occur especially at higher age such as neurodegenerative diseases and cancer. Caloric restriction (CR) promotes longevity and healthy ageing. Currently, the contributing role of autophagy in the context of CR-induced health benefits is being unravelled. Furthermore recent studies imply that the advantages from polyphenol consumption may be also connected to autophagy induction. In this review, the literature on autophagy regulation by (dietary) polyphenols such as resveratrol, catechin, quercetin, silibinin and curcumin is discussed with a focus on the underlying molecular mechanisms. Special attention is paid to the implications of age-related autophagy decline for diseases and the possibility of dietary countermeasures.
Astrocyte swelling is an integral component of cytotoxic brain edema in ischemic injury. While mechanisms underlying astrocyte swelling are likely multifactorial, oxidative stress and mitochondrial dysfunction are hypothesized to contribute to such swelling. We investigated the protective effects of cinnamon polyphenol extract (CPE) that has anti-oxidant and insulin-potentiating effects on cell swelling and depolarization of the inner mitochondrial membrane potential (ΔΨm) in ischemic injury. C6 glial cells were subjected to oxygen-glucose deprivation (OGD) and cell volume determined using the 3-O-methyl-[3H]-glucose method at 90 min after the end of OGD. When compared with controls, OGD increased cell volume by 34%. This increase was blocked by CPE or insulin but not by blockers of oxidative/nitrosative stress including vitamin E, resveratrol, N-nitro-L-arginine methyl ester (L-NAME) or uric acid. Mitochondrial dysfunction, a key component of ischemic injury, contributes to cell swelling. Changes in ΔΨm were assessed at the end of OGD with tetramethylrhodamine ethyl ester (TMRE), a potentiometric dye. OGD induced a 39% decline in ΔΨm and this decline was blocked by CPE as well as insulin. To test the involvement of the mitochondrial permeability transition (mPT), we used Cyclosporin A (CsA), an immunosuppressant and a blocker of the mPT pore. CsA blocked cell swelling and the decline in ΔΨm but FK506, an immunosuppressant that does not block the mPT, did not. Our results show that CPE reduces OGD-induced cell swelling as well as the decline in ΔΨm in cultures and some of its protective effects may be through inhibiting the mPT.