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Cholecalciferol (vitamin D3) is a
fat-soluble vitamin, which is widely used for the prevention and treatment of
vitamin D deficiency. The aim of the study was to investigate the impact of the
Trivedi Effect®-Consciousness Energy Healing Treatment on the
isotopic abundance ratios (PM+1/PM and PM+2/PM)
along with the structural properties of vitamin D3 using advanced
spectroscopy methods. Vitamin D3 sample was divided into two parts,
one part of the sample was termed as a control sample, while the other part of
the sample received the Trivedi Effect® (Biofield Energy Treatment)
remotely by a famous Biofield Energy Healer, Mr. Mahendra Kumar Trivedi termed
as the Biofield Energy Treated sample. The liquid chromatography-mass
spectrometry (LC-MS) chromatograms of both the cholecalciferol samples showed a
single largest peak at the retention time (Rt) 20.6 min and
protonated molecular mass peak at m/z
385.3 (calcd for C27H45O+, 385.35) in the mass
spectra. The LC-MS based isotopic abundance ratios of PM+1/PM
(2H/1H or 13C/12C or 17O/16O)
and PM+2/PM (18O/16O) were
significantly increased by 15.20% and 10.44%, respectively in the treated
cholecalciferol compared to the control sample. Thus, the 13C, 2H
and 17O contributions from C27H45O+
to m/z 386 and 18O
contribution from C21H21O6+ to m/z 387 in the treated sample was
significantly increased compared with the control sample. The gas
chromatography-mass spectrometry (GC-MS) spectral data showed that the
molecular mass peak intensities (m/z
384.4) in the treated cholecalciferol at Rt 23.28 and 23.88 min were
increased by 4.54% and 3.21%, respectively compared with the control sample.
The proton and carbon signals for CH3, CH2, CH, C-OH and
=C= groups in the 1H and 13C Nuclear Magnetic Resonance
(NMR) spectra of the treated and control samples were similar. The improvement
in the isotopic abundance ratios and mass peak intensities of the treated
cholecalciferol might be due to the possible mediation of neutrinos via the
Trivedi Effect®-Consciousness Energy Healing Treatment. The
increased isotopic abundance ratio of the treated cholecalciferol might have a
stronger atomic bond, increase the stability and alter the rate of metabolic
reactions in the body. Thus, the Biofield Energy Treated cholecalciferol would
be more efficacious nutraceutical and pharmaceutical formulations which might
provide better therapeutic response counter to deficiency of vitamin D,
rickets, osteoporosis, diabetes mellitus, cancer, cardiovascular diseases,
infections, etc.
Keywords: Cholecalciferol, The Trivedi Effect®, Energy of
consciousness healing treatment, LC-MS, Isotopic abundance, GC-MS, Kinetic
isotope effects
INTRODUCTION
Cholecalciferol
(vitamin D3) is found in foods and also in the dietary supplement to
overcome vitamin deficiency and associated disease [1]. It has multiple effects
on the human body, which regulate the functions of muscles, brain, lungs,
liver, kidneys, heart, immune system, pancreas, large and small intestines.
Vitamin D receptors are ubiquitously found in most of the body parts. Vitamin D
receptor response elements with hundreds of genes directly or indirectly
influence cell-to-cell communication, normal cell growth, cell cycling and proliferation,
cell differentiation, maintenance of calcium and phosphorus balance, hormonal
balance, neurotransmission, skin health, immune and cardiovascular functions
[1-3]. Deficiency of vitamin D occurs in those who
have an insufficient dietary intake or who
fail to produce enough vitamin D3 in their skin from its precursor,
7-dehydrocholesterol, in response to exposure to ultraviolet light. Vitamin D
deficiency conditions also caused by intestinal malabsorption or chronic liver
disease, familial hypophosphatemia, and for the hypocalcaemia that is
associated with hypoparathyroidism [2]. Vitamin D shortage in the body plays a
critical role in several diseases, e.g. rickets, osteoporosis, arthritis,
multiple sclerosis, cancer, diabetes mellitus, mental disorders, cardiovascular
diseases, infections, cognitive impairment in older adults, Parkinson’s and
Alzheimer’s diseases, dementia, glucose intolerance, multiple sclerosis, etc.
[3-6]. In the USA, 15 µg/d (600 IU per day) is required for all individuals
between all the age group [7]. High dose vitamin D supplementation may cause
toxicity like hypercalcemia, polyuria, polydipsia, weakness, mental retardation
and insomnia [8]. Vitamin D stability is more concerned as it is a heat and
light-sensitive compound [9,10]. The transformation mechanism of vitamin D and
the absorption of its active form (vitamin D3) are very complicated.
Vitamin D3 bioavailability directly affected by various factors such
as dietary fiber, genetic factors and the status of vitamin D3 [11].
Recent
studies revealed that the bioavailability profile of several
pharmaceutical/nutraceutical compounds, i.e., 25-hydroxyvitamin D3
[25(OH)D3], resveratrol, berberine, etc. are significantly altered
by means of the Trivedi Effect®-Consciousness Energy Healing
Treatment [12-14]. The “Biofield Energy” is a type of electromagnetic energy
field generated by continues moment of the charged particles (i.e., cells, ions,
etc.) in the human body [15,16]. The Biofield Energy Healers has the ability to
harness the energy from the “Universal Energy Field” and can transfer into any
object(s) around the globe. The process of treatment to an object is known as
the Biofield Energy Healing Treatment. There are several Energy Therapies that
are used nowadays against various disease conditions [17,18]. The Energy
Therapy has been recognized worldwide as a Complementary and Alternative
Medicine health care approach by the National Center of Complementary and
Integrative Health with other therapies, medicines and practices such as Qi
Gong, Tai Chi, Ayurvedic medicine, traditional Chinese herbs and medicines,
aromatherapy, meditation, yoga, chiropractic/osteopathic manipulation,
acupressure, homeopathy, acupuncture, healing touch, hypnotherapy, movement
therapy, Reiki, cranial sacral therapy, etc. [19]. The Trivedi Effect®
has surprizing ability to transform the characteristic properties of metals and
ceramic [20, 21], organic compounds [22,23], nutraceuticals [24],
pharmaceuticals [25,26], culture medium [27,28] and improve the overall
productivity of crops [29], alteration of the isotopic abundance ratio in the
organic compounds [30-32] may be through the possible mediation of neutrinos
[15].
There
are wide applications of study on the natural stable isotope ratio analysis in
several fields of sciences to understand the isotope effects resulting from the
alterations of the isotopic composition [33-35]. Gas chromatography-mass
spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), are
widely used for the analysis of isotope ratio with sufficient precision [34].
Thus, the isotopic abundance ratio analysis of PM+1/PM (2H/1H
or 13C/12C or 17O/16O) and PM+2/PM
(18O/16O) was performed to evaluate the influence of the
Trivedi Effect® on the isotopic abundance ratio in cholecalciferol.
Similarly, the LC-MS, GC-MS and NMR (Nuclear Magnetic Resonance) techniques
were also used to characterize the structural properties of cholecalciferol.
MATERIALS AND METHODS
Chemicals and reagents
The
test sample cholecalciferol (>98%) was purchased from Sigma-Aldrich, India
but other chemicals used during the experiments were of analytical grade
purchased in India.
Consciousness energy healing
treatment strategies
The
test sample cholecalciferol was divided into two equal parts. One part of the
test cholecalciferol was considered as a control sample, which was not received
the Biofield Energy Treatment. Whereas, the second part was received the
Consciousness Energy Healing Treatment (Trivedi Effect® for 3 min)
remotely under standard laboratory conditions and known as treated
cholecalciferol. This Biofield Energy was provided through the healer’s unique
energy transmission process by a famous Biofield Energy Healer, Mr. Mahendra
Kumar Trivedi (USA), to the test item. The control cholecalciferol was treated
with a “sham” healer, who did not have any knowledge about the Biofield Energy
Treatment. After the treatment both the samples of cholecalciferol were kept in
sealed conditions and characterized.
Characterization
Liquid chromatography-mass
spectrometry (LC-MS) analysis and calculation of isotopic abundance ratio: The LC-MS analysis of the samples was carried out with the
help of LC-Dionex Ultimate 3000, MS-TSQ Endura, USA equipped with a photo-diode
array (PDA) detector connected with a triple-stage quadrupole mass spectrometer
(Thermo Scientific TSQ Endura, USA) with a Thermo Scientific Ion Max NG source
and Atmospheric Pressure chemical ionization (APCI). The analysis was performed
on a reversed phase Zorbax SB-C18 100 × 4.6 mm, 3.5 µm in gradient mode in the
liquid chromatograph (column temperature 40°C). The mobile phase was ammonium
formate and 0.5% formic acid in water (A) and acetonitrile (B) at a constant
flow rate of 0.6 mL/min. The injection volume was 10 µL and the total run time
was 30 min. Chromatographic separation was achieved using gradient condition as
follow: 0 min-50% B, 5 min-90% B, 10 min-100% B, 20 min-100% B, 25 min-50% B
and 30 min-50% B. Peaks were monitored using the PDA detector. The mass
spectrometric analysis was performed under +ve ESI mode.
The
natural abundance of C, O and H isotope can be predicted from the comparison of
the relative abundance of the isotope peak with respect to the base peak. The
values of the natural isotopic abundance of the common elements are obtained
from the literature [36-39]. The change in the isotopic abundance ratios (PM+1/PM
and PM+2/PM) for the control and treated cholecalciferol
was calculated using equation (1).
% Change in isotopic abundance ratio = [(IARTreated
– IARControl) / IARControl) × 100]
(1)
Where
IARTreated and IARControl is the isotopic abundance ratio
in the treated and control cholecalciferol, respectively.
Gas chromatography-mass
spectrometry (GC-MS) analysis: An Agilent 7890B GC equipped
with a silica capillary column HP-5 MS (30 m × 0.25 mm × 0.25 µm) and coupled
to a quadrupole detector with pre-filter (5977B, USA) was operated with
electron impact (EI) ionization in positive mode at 70 eV. The oven temperature
was programmed from 50°C (1 min hold) to 150°C @ 20°C/min to 200°C (6 min hold)
@ 25°C/min to 280°C @ 20°C/min (12 min hold). Temperatures of the injector,
detector (FID), auxiliary, ion source and quadrupole detector were 230, 250,
280, 230 and 150°C. Cholecalciferol was dissolved in methanol, and 5.0 µL was
splitlessly injected with helium as a carrier gas with a flow rate of 2.0
mL/min.
The
percent change in peak intensity (I) was calculated using the following
equation (2):
% Change in peak intensity (I) = [ITreated – IControl]/IControl × 100 (2)
Where,
IControl and ITreated are the peak intensity of the
control and Biofield Energy Treated sample, respectively.
Nuclear magnetic resonance
(NMR) analysis: 1H NMR spectra of cholecalciferol
were recorded at at 400 MHz MHz on Agilent-MRDD2 FT-NMR. Approximately 3 mg of
the sample was dissolved in DMSO-d6. Chemical shifts (d) were in parts per
million (ppm) relative to the solvent’s residual proton chemical shift {(CD3)2SO,
δ=2.5}. Similarly, 13C NMR spectra of cholecalciferol were measured at at 100
MHz on Agilent-MRDD2 FT-NMR spectrometer spectrometer at room temperature. The
sample was dissolved in DMSO-d6. The solvent’s residual carbon chemical shift
{(CD3)2SO, δ=39.52}.
RESULTS AND DISCUSSION
Liquid chromatography-mass
spectrometry (LC-MS) analysis and isotopic abundance ratio analysis
The
control and treated cholecalciferol showed a sharp chromatographic peak at the
retention times (Rt) of 20.63 and 20.62 min, respectively (Figure
1). The % peak area at Rt 20.6 min was 99.58 and 99.54 in
control and Biofield Energy Treated sample, respectively. This indicated that
the polarity of the Biofield Energy Treated sample remained similar compared to
the control cholecalciferol.
The mass spectra of the control and treated
samples at Rt of 20.6 min exhibited the presence of the molecular
ion of cholecalciferol (C27H45O+) adduct with
hydrogen ion (Figure 2) at m/z 385.3 (calcd for C27H45O+,
385.35). The lower m/z showed the
presence of the [M-OH]+ ion mass peak at m/z
367.3 (calcd for C27H43+, 367.3) in both the cholecalciferol
samples (Figure 2).
The mass spectrum of the Biofield Energy Treated cholecalciferol showed
an almost similar type of mass fragmentation pattern of the control sample (Figure
2). The molecular ion peak at m/z
385.3 exhibited 100% relative peak intensity in both the mass spectra (Figure
2). The relative peak intensities of the other ion peaks in the
Consciousness Energy Healing Treated cholecalciferol were significantly altered
compared to the control sample.
The control and treated samples of cholecalciferol showed the mass of a
protonated molecular ion at m/z 385.3
(calcd for C27H45O+, 385.35) with 100%
relative abundance in the mass spectra. The theoretical calculation of isotopic
peak PM+1 for the protonated cholecalciferol presented as below:
P (13C) = [(27 × 1.1%) × 100% (the actual size of the M+
peak)] / 100% = 29.7%
P (2H) = [(45 × 0.015%) × 100%] / 100%= 0.675%
P (17O) = [(1 × 0.04%) × 100%] / 100% = 0.04%
PM+1, i.e., 13C, 2H and 17O
contributions from C27H45O+ to m/z 386 = 30.42%
Similarly, the
theoretical calculation of isotopic peak PM+2 for the protonated cholecalciferol
presented as below:
P (18O) = [(1 × 0.20%) x 100%] / 100% = 0.2%
PM+2 of 18O contribution from C27H45O+ to m/z 387 = 0.2%
The calculated isotopic abundance of PM+1 value 30.42% was higher to
the observed value (23.69%), but the calculated PM+2 value 0.2% was lower to the observed value (3.64%) (Table 1). The probability of A+1
and A+2 elements having an isotope
with one and two mass unit heavier, respectively than the most abundant isotope
(i.e., 13C, 2H, 17O and
18O) contributions to the
mass of the isotopic molecular ion [M+1]+ and [M+2]+.
2H did not contribute much any isotopic m/z ratios because of its less natural abundance compared to the
natural abundances of C and O isotopes [37,38]. From the calculations, it was
observed that 13C, 17O and 18O have the major
contributions from cholecalciferol to the isotopic mass peak at m/z 386 and 387. Therefore, PM, PM+1
and PM+2 of the
cholecalciferol at m/z 385, 386 and
387 of the control and Biofield Energy Treated samples were
obtained from the experimental relative abundance of M +, (M+1)+
and (M+2)+ peaks, respectively in the mass spectra (Table 1).
The isotopic abundance ratio of PM+1/PM
(2H/1H or 13C/12C or 17O/16O)
in the Trivedi Effect®-Consciousness Energy Healing Treated
cholecalciferol was significantly increased by 15.20% compared to the control
sample (Table 1). Thus, the 13C,
2H and 17O contributions from C27H45O+
to m/z 386 in the Biofield Energy
Treated sample was significantly increased compared to the control sample.
Similarly, the isotopic abundance ratio of PM+2/PM (18O/16O) in the Biofield Energy Treated
cholecalciferol was significantly increased by 10.44% compared to the control
sample (Table 1). Thus, the 18O
contribution from C27H45O+ to m/z 387 in the Biofield Energy Treated
cholecalciferol was also significantly increased compared to the control
sample.
Neutrons and alteration in its number in the
molecule lead to the increased or decreased isotopic abundance of the
compounds. The changes in atomic/molecular weights are postulated to the
changes in atomic mass and charge via
the possible mediation of neutrinos [15,40-42]. The recent innovation of
neutrino oscillations seems to give credence to the postulates of Mr. Mahendra
Kumar Trivedi on the Trivedi Effect® [15]. Thus, it can be assumed
that the Trivedi Effect®-Consciousness Energy Healing Treatment might
be providing the necessary energy for the neutrino oscillations leads to the
modification of the fundamental physicochemical properties of a compound
[43,44]. The increased isotopic abundance
ratios 2H/1H or 13C/12C or 17O/16O
or 18O/16O would highly
influence the atomic bond vibration and increase bond strength of treated cholecalciferol
[45]. The alteration in the isotopic
abundance ratio of the atoms/molecules cause of the change in the kinetic
isotope effects, which is very useful to study the reaction mechanism,
understand the enzymatic transition state, and enzyme mechanism that is
supportive for designing effective, and specific inhibitors, etc. [34].
Therefore, The Biofield Energy Treated cholecalciferol with improved
isotopic abundance ratio (PM+1/PM and PM+2/PM)
might be advantageous for the better nutraceutical and pharmaceutical
formulations.
Gas
chromatography-mass spectrometry (GC-MS) analysis
The GC-MS chromatograms of the control and Biofield Energy Treated
cholecalciferol showed two clear independent peaks (Figure 3). The Rt
of the control sample was at 23.28 and 23.88 min, whereas Biofield Energy
Treated sample at 23.28 and 23.88 min showed that the Rt of both the
sample is very close. Therefore, the results indicated that the polarity of the
Biofield Energy Treated cholecalciferol remained close compared to the control
sample. The chromatograms of both the control and Biofield Energy Treated
showed two peaks might be due to the cis and
trans isomers of cholecalciferol [46,47].
The GC-MS spectra of the control and treated samples at Rt of
23.28 min exhibited the presence of the molecular ion of cholecalciferol (C27H44O+)
(Figure 4) at m/z 384.4 (calcd
for C27H44O+, 384.34). The lower mass
fragmentation peak at m/z 366, 351.4
and 325.3 for C27H42•+, C26H39••+
and C24H37+, respectively in both the spectra (Figure
4). The mass fragmentation pattern of the Biofield Energy Treated
cholecalciferol was similar to that of the control sample. But the mass peak
intensities of the treated cholecalciferol were altered compared to the control
sample. The mass peak intensity of the control and treated cholecalciferol were
1384814.88 and 1447749.88, respectively at Rt of 23.28 min.
Similarly, the mass peak intensities of the control and Biofield Energy Treated
cholecalciferol were 1219705.00 and 1258803.38, respectively at Rt
of 23.88 min. The mass peak intensities at Rt 23.28 and 23.88 min in
the Biofield Energy Treated sample were increased by 4.54% and 3.21%,
respectively compared to the control sample (Table 2). The mass peak
intensities were significantly increased which might be possible due to the
impact of the Trivedi Effect®-Consciousness Energy Healing
Treatment. The Trivedi Effect® is a proved natural phenomenon which
has the remarkable potential to alter the isotopic abundance ratios of various
compounds through the possible mediation of neutrinos [15,43,44].
Nuclear
magnetic resonance (NMR) spectroscopy analysis
Figures 5 and 6 showed the
1H, and 13C NMR spectra, respectively for the control and
Biofield Energy Treated cholecalciferol. The analyzed NMR spectral data of both
the samples are presented in Table 3.
The 1H NMR spectra of both the samples indicated that signals for
the protons coupling of CH3, CH2, CH and OH protons of
cholecalciferol were in the range of d 0.48 to
6.19 ppm (Figure 5 and Table 3),
which were very close to each other. Similarly, the carbon signals for CH3,
CH2, CH, =C= and C-OH groups in the 13C NMR spectrum (Figure 6) were in the range of
11.74-145.45 in both the control and Biofield Energy Treated samples of
cholecalciferol (Table 3). The
experimental results were closely matched to the reported literature [48]. The 1H
and 13C NMR spectral data concluded that there was no structural
modification of the treated cholecalciferol compared to the control sample.
CONCLUSION
The present
experimental data suggest that the Trivedi Effect®-Energy of Consciousness Healing
Treatment has shown a significant impact on the isotopic abundance ratios and
relative peak intensities of cholecalciferol/vitamin D3. The LC-MS
chromatograms of both the cholecalciferol samples showed a single largest peak
at the retention time (Rt) 20.6 min and protonated molecular mass
peak at m/z 385.3 (calcd for C27H45O+,
385.35) in the mass spectra. The LC-MS
based isotopic abundance ratios of PM+1/PM (2H/1H
or 13C/12C or 17O/16O) and PM+2/PM
(18O/16O) were
significantly increased by 15.20% and 10.44%, respectively in the Consciousness Energy Healing Treated
cholecalciferol compared to the control sample. Thus, the 13C,
2H, and 17O contributions from C27H45O+
to m/z 386 and 18O
contribution from C21H21O6+ to m/z 387 in the Consciousness Energy Healing Treated cholecalciferol were
significantly increased compared with the control sample. The GC-MS spectral
data showed that the molecular mass peak intensities (m/z 384.4) in the Consciousness
Energy Healing Treated cholecalciferol at Rt 23.28 and 23.88 min were increased by 4.54% and 3.21%, respectively compared with the
control sample. The improvement in the
isotopic abundance ratios and mass peak intensities of the Consciousness Energy
Healing Treated cholecalciferol might be due to the possible mediation of
neutrinos via the
Trivedi Effect®-Consciousness Energy Healing Treatment. The increased isotopic abundance ratio of the Consciousness Energy Healing Treated cholecalciferol might have a stronger atomic bond,
increase the stability, and alter the rate of metabolic reactions in the body.
Thus,
the Trivedi Effect®-Consciousness
Energy Healing Treated vitamin D3 would be more advantageous to develop
more efficacious nutraceutical/pharmaceutical formulations which might provide
better therapeutic response against vitamin D deficiency, rickets,
osteoporosis, arthritis, diabetes mellitus, multiple sclerosis, cancer,
cardiovascular diseases, inflammations, infections, mental disorders, stress,
aging, glucose intolerance, Parkinson’s and Alzheimer’s diseases, dementia,
cognitive impairment in older adults, etc.
ACKNOWLEDGEMENT
The authors are grateful to GVK Biosciences
Pvt. Ltd., Trivedi Science, Trivedi Global, Inc., Trivedi Testimonials and
Trivedi Master Wellness for their assistance and support during this work.
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Table 2
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Table 3
