JOURNAL PRESENTATION

Dr Deepa J G
IIIyr ,P G Scholar Dept of Panchakarma
TITLE - Plasma metabolomics reveal the
correlation of metabolic pathways and Prakritis of
humans
• Original Research Article
• Authored by : Amey Shirolkar , Sutapa Chakraborty , Tusharkanti Mandal,
Rajesh Dabur
• Received: 2/10/2016 Accepted:12/05/2017 Published: 26/11/2017
• DOI: 10.1016/j.jaim.2017.05.002
• Journal : Journal of Ayurveda and Integrative Medicine
• Impact Factor: 0.83
RELEVANCE
• Sukraartavasthaou janmadou visheneva vishakrime|
taischa tisra prakrutayo hIna midhyottama pruthak|
sama dhatu samastasu sreshta nindya dwidoshaja|| (AH/Su/1)
• Prakruti is a powerful tool in hands of Ayurveda but least explored
one
• Previous studies validates and put forth questionnaires for analysis
• Knowing prakruti helps in prevention and curation of a dz easy
• Prakruti analysed in terms of metabolomics may contribute a new
scientific outlook to the existing knowledge of Prakruti
INTRODUCTION
• Panchabhuta combine in pairs to form three dynamic forces/
interactions called ‘doshas’ which governs principles of living beings
• All doshas are present in every human being; however, only one is
dominant. Ie their prakruti
• Vata refers to functions of motion; Pitta - digestion & metabolism;
Kapha -anabolic activity & accumulation
• Functions are closely related to metabolic events in body and hence
to phenotypic expressions
• Therefore metabolome of an individual can be directly correlated
with Prakriti
• This study, metabolomic approach was used to identify the dominant
metabolic pathways of different dominating Prakritis.
OBJECTIVES
• Analysis of plasma metabolites and related pathways to
corelate Prakriti specific dominant marker metabolites and metabolic
pathways
• Analysis of Prakriti of individuals via Ayurveda
• Collection of plasma and analysis of metabolites in each Prakriti
• Identifying the dominant marker metabolites representing each Prakriti
• Analysing metabolic pathways and arrive at valid conclusions
Study Design
• Study type : Corelational research
• Phases of study : Phase 1 – clinical & Phase 2 - analytical
• H1: Analysis of plasma metabolites and related pathways helps to
corelate Prakriti (specific dominant marker metabolites and metabolic
pathways)
• H0 : Analysis of plasma metabolites and related pathways doesn’t
help to corelate Prakriti (specific dominant marker metabolites and
metabolic pathways)
• Study Site: PDDYP Ayurveda College, Pune
Materials and Methods
205 subjects were screened and 38 healthy males were included in the study
• Inclusion criteria: Healthy males free from Diabetes, hyperlipidemia,
hypertension & other chronic systemic disorders
• Exclusion Criteria : Smoking and alcohol habits
Materials : Ethics Committee approval, CTRI regtn, informed consent
Prakriti analysis self validated questionnaire
 Fasting blood plasma samples
HPLC (mobile – solvent and stationary phase- sample), Solvents used for
sample processing and in LCMS assembly: acetonitrile, methanol, formic
acid and water were of LC–MS grade
 Standards of metabolites and Cytoscape for metabolic pathway identfctn
 I N T E R V E N T I O N
PHASE 1
Clinical
Enrolment
Inclusion of 38 males
Prakriti analysis using self framed
questionnaire and by 2 Ayur Doctors
Asked to follow vegetarian diet and
restrain from medicine a week prior to an assembly of an assembly of C18 ZORBAX
blood test
On 7th Day – fasting blood samples
were collected, centrifuged and
separated
Lypholised samples were made
Phase II – analytical phase
HPLC
High Pressure Liquid chromatography
2 systems were used
Stationary phase: Solvent
(A) - 0.1% formic acid–water and (B) 0.1%
formic acid–acetonitrile
Mobile phase : 15 microlit of each sample into
(4.6 × 12.5 mm) guard column followed by
ZORBAX 300SB-C18 (4.6 × 150 mm)
Use: To separate & identify different
metabolites in each sample
(HPLC-Q-TOF-MS)High pressure liquid
chromatography coupled with quadrupole time-of-
flight mass spectrometry
Operated in + ion polarity
ionization type electrospray ionization (ESI),
gas temperature 350 °C, nebulizer 45 psi, gas
flow 8 L/min ,fixed collision energy of 25.0 V
and acquisition rate 4 spectra per cycle.
HPLC-Q-TOF-MS technique enables idntfn of
unknown compound based on prediction of
chemical formula from accurate ion mass
measurement and characteristic isotopic
pattern .The volume of sample needed for
analysis is very small .Its a powerful tool for
idntfn of species and can be very useful when
reference standards arent available.
HPLC-Q-TOF-MS identify metabolite – it
mass structure …
Metabolic Pathways were identified
using cystoscape and its softwares
ANALYSIS
• Multivariate analysis: Mass Hunter Qualitative software analysis – for
preliminary quality check
• Profinder software: for baseline correction, noise reduction,
background contaminants removal and extraction of molecular
features having specification of 5000 rpm, 5ppm etc were extracted
• Mass Profiler Professional software used for alignment and
compounds present in <75% samples were removed
• Data is used to generate Box- Whisker plot and Principal Component
Analysis, Partial least-squares-discriminant analysis (PLS-DA), a class
prediction algorithms was performed to distinguish 3 Prakriti groups
and explore unique features present across the groups
RESULT
• HPLC result says average of 23 peak containing 2000 compounds and
extracted Base Peak Chromatograms shows V-P-K distinct peaks
• Only the compounds present universally in >75% of samples in each
group were extracted for further processing to ensure the consistency
of metabolomics profiles in each group.
• After statistical analysis 76 metabolites were arranged in K, P, and V.
• 76 Differentially expressed metabolites were compared
(fragmentation pattern)with standards in human metabolome in
Metlin library, HMDB and NIST databases
Vata metabolites Pitta metabolites
Omega Fatty acid oxidation metabolites A steroid intermediate, Bile salt is more
are more
Diglyceride is more which prevent fat 6/11 amino acid metabolites are
accumulation in body present
valine, leucine and isoleucine
degradation is more (in stress, energy
and muscle metabolism,)
Out of 4 vit D derivative max D3 is in P
hexanoylcarnitine produce metabolic
error is present
melatonin, an antioxidant, nucleic acid
protector and regulator of body clock
guanidoacetic acid, a creatine
precursor that causes cardiovascular
and skeletal problems
Kapha metabolites
A Vasodialator and platelet aggregatn
factor is present
Fatty acid oxidation disorder Metabolite
is more
arginino-succinic acid, AA in Kapha is
asso with removal of N2 [urea cycle
]from body
Carnitines are more in K [ fatty acid to
mitochondria for energy]
Sphinganine, inhibit LDL
D-threitol,an indicator of carbohydrate
metabolism derangements
11 unidentified metabolites are seen
Vata prakriti metabolic pathway
Vata Prakriti cellular detoxification
process along with neurotransmitters i.e.
dopamine [feel good hormone]and
glutamate []learning] was found to be
dominant
These processes are important to excrete common, may be because of increased
waste and cognition
Shows upregulated arachidonic acid
[inflmatry or cognitve imprmnt reponse],
beta-alanine[lean muscle] catecholamine
[fli-fright chala] and glycerolipid
metabolism [lipid catabolism]
Vata individuals may have predisposition
to neurological problems and insulin
resistance
Pitta prakriti metabolic pathway
Pitta Prakriti metabolism of xenobiotics
/drug and toxins was observed through
cytochrome P450 [toxin removal]
Beta-oxidation disorders are more
lipid absorption through the intestine as
increased levels of bile acids were
observed
Melatonin and 2-methoxyestrone,
hormones to maintain circadian
biological clock, basal metabolism rate,
and physical constitution, were found to
be dominant
All these processes can be correlated
with fast metabolism of pitta Prakriti
Kapha prakriti metabolic pathway
Predisposed to respiratory problems
[ cellular resp]
•Continuous mitochondrial genome
maintenance is the dominant bioprocess
• Upregualted NAD pathway [ageing]
• pyrimidine, membrane lipid
Says slow catabolism of lipids
Leads to lipid storage and weight gain
Increased lipids increase oxidative stress
and CVD
Lipid metabolism markers like LDL, TC, TG
&neurological dz markers r more
Fc gamma R mediated phagocytosis –
seen in immunity
STATISTICS – correlational design does not imply
correlation analysis
• Multivariate analysis of plasma metabolome is done to establish the correlation between
metabolomes and prakriti
• Regression value R =0.97 is used  correlation study says very high corelation
• Data after MS is subjected to one way ANOVA (using MPP) with permutative Benjamin–Hochberg
FDR correction and Tukey HSD post hoc to minimize the FDR, keeping p value cutoff < 0.05 and
fold change > 2.0 {for p value cutoff < 0.05, false discovery rate (FDR) become <5%}.
• Box and whisker plot: Descriptive statistics, a method for graphically depicting groups of
numerical data through their quartiles
• Principal component analysis (PCA) is a statistical procedure that uses an orthogonal
transformation to convert a set of observations of possibly correlated variables to principal
components
• Partial least squares regression (PLS regression) is a statistical method that bears some relation
to principal components regression
• In pathway analysis, q<0.01 is taken
 DRAW BACKS –
a) Kapha Vata, Pitta Vata
prakrutis are not considered
or included ??
b) Allocation details of
38/205 is nt stated
LIMITATIONS OF
STUDY
1. Gender bias in inclusion
criteria
2. Self formulated Prakruti
analysis tool – not validated ?
3. Study population is small
RELATED ARTICLES
• Patwardhan B., Joshi K., Chopra A. Classification of human population
based on HLA gene polymorphism and the concept of Prakriti in
Ayurveda. J Altern Complement Med. 2005;11(2):349–353.[PubMed]
Lessons learnt
• Scope of development of Ayurmetabolomics
• Data about simple metabolites can classify human phenotypes
• Predicts disease that persons are prone
• Prognosis of the disease
• Prevents the disease onset
THANK YOU ....