Metabolomics

La metabolomica
nella diagnosi di sepsi
Vassilios Fanos
Neonatal Intensive Care Unit, Neonatal Pathology,
Puericulture Institute and Neonatal Section
AOU and University of Cagliari
Agenda
Agenda
The Evolution which
becomes Revolution (?)
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Background (the new languages of Medicine)
What actually is metabolomics?
Clinical metabolomics in Neonatology
Metabolomics and Infections (Experimental studies, Clinical
studies in: Newborn, Child, Adult)
 Conclusions (Individualized Medicine)
I nuovi linguaggi della Medicina
 Biologia dei sistemi
 Medicina dei sistemi
 Medicina delle reti
 Tecnologie «omiche»
 Medicina trainata dai dati
 Big data in Medicina
 Resilienza, antifragilità
 Medicina olistica
 Medicina personalizzata
Parola chiave: complessità
2
0
1
2
More Omics Take Off: Metabolomics
More Omics Take Off: Metabolomics (mod.)
Metabolomics = Metabonomics
•Metabolism and metabolomics share as their root the ancient Greek
word, metabol, which means change, and obviously both terms are
equally applicable to cell, tissue or whole organism
•The total repertoire of small molecules present in cells, tissues,
organs, and biological fluids
• Sugars
• Lipids
• Small peptides
• Vitamins
• Amino acids
Metabolomics = Metabonomics
•Metabolism and metabolomics share as their root the ancient Greek
word, metabol, which means change, and obviously both terms are
equally applicable to cell, tissue or whole organism
•The total repertoire of small molecules present in cells, tissues,
organs, and biological fluids
• Sugars
• Lipids
• Small peptides
• Vitamins
• Amino acids
August
2010
«The escalator»
April 2012
“a priori”: hypothesis testing
“a posteriori”: hypothesis generating
HOW
does it
work?
ScaleFree
Networks
(hub and
spoke)
Urine
NMR spectroscopy
GC-MS (Gas Cromath.-Mass Spectrometry)
By Saude 2010 3 Dimensions
A 1H NMR-BASED METABOLOMIC STUDY OF URINE
IN CHILDREN WITH RENAL DISEASES
2 Dimensions
Atzori L, Agostiniani, Cortesi P, Antonucci R.,… Fanos V.
Frontiers in Bioscience 2010
Among
hundreds of
metabolites
VIP (variable
influence the
proiection)
are selected
Healthy newborn
1H-NMR urine sample
Newborn with renal disease
1H-NMR urine sample
VIP VARIABLES: Hyppurate, tryptophane, phenylalanine, malate,
tyrosine, hydrossibutirrate, N-acetil-glutammate, tryptophan, proline
Metabolomic pathways in Autism (unpublished data)
Fanos V. in collaboration with R. Francavilla (Bari)
Harvard Magazine, April 2014
Modern analytical technologies allow for the identification
of patterns that confer significantly more information than
the measurement of a single parameter, much as
a bar code contains more
information than a single number
metabolomics and sepsis……………… 41 papers
metabolomics and neonatal sepsis…..7 papers
7
studies
for the
future
(mod.)
accuracy
over 94%
Non survivors
septic rats
Controls
Survivors
septic rats
Lung tissue
BALF
Serum
SENSITIVITY 100%, SPECIFICITY 100%
J Chemother October 2013
Metabolomics: a gold standard for the diagnosis
of neonatal sepsis?
•Fanos V (Cagliari)
•Corsello G (Palermo)
•Stronati M (Pavia)
•Gazzolo D (Alessandria)
198,500 euros
Control
GC-MS.
A comparison between the GC-MS
chromatograms of urine samples
collected from control and septic
newborns
Septic
newborn
it was possible to identify the metabolites responsible
for the differences between septic neonates and controls:
for EOS neonates at the time of birth and for the LOS neonates
within 72 hours before the clinical onset
Control
Septic
newborn
Individual metabolites
discriminating were:
 mannitol,
 4-hydroxyphenylacetate,
 p-cresol,
 myo-inositol,
 trimethylamine-Noxide
 1methylnicotinamide.
JPNIM, 2012, open access
CMV
INFECTION
AT BIRTH
A
PLS-DA scores plot of control (filled circles)
vs asymptomatic (empty circles) urine
samples.
Fig. 1 Aliphatic region of the 1H NMR spectra of urine samples from (a) control and (b) CMV asymptomatic subjects.
Diagnosing Pneumonia in children
Urine metabolomics
Laiakis EC et al PLoS one, 2010
Diagnosing pneumonia with urinary metabolomics
(Future Medicine 2010)
Monitoring pneumonia in children
(urinary metabolomics)
Septic
Healthy
controls
SIRS
Loss of ATP
homeostasis
Apoptosis
(Ptd =
phosphatidylserine )
Disruption
of
endothelial
barrier
function
Oxydative
stress
Metabolites markers of severity of infection
Possible associations between
acute physiology score (APS) and
myoinositol (A) and total
glutathione (B). The associations,
suggest that myoinositol (ρ =
0.53; P = 0.05; q = 0.25) and total
glutathione (ρ = 0.56; P = 0.04; q =
0.25) may be markers of sepsisinduced ALI severity.
2014
ICU controls (n = 20):
diamonds
cutoff line
Septic shock samples
(n = 39): gray
triangles
March 2014
JANUARY 2014, BOSTON GROUP
Relationship of 5 metabolites included in Bayesian Network to 28-day mortality.
This network achieves 91% AUC.
Directed edges indicate statistical dependence and do not represent causative links.
Metabolomics in
pneumonia and sepsis
Patent WO 2014004539 A1
ipo di pubblicazione
Richiesta
Numero domanda PCT/US2013/047662
Data di pubblicazione
3 gen 2014
Data di registrazione
25 giu 2013
Data di priorità
26 giu 2012
Inventori John A. Kellum, Christopher W. SEYMOUR
Candidato
University Of Pittsburgh-Of The
Commonwealth System Of Higher Education
3 metaboliti strategici
A questo punto, la predittività è possibile…
Ma tutto questo è etico?
2010
A
Asystole group (BLACK CIRCLE)
Non-Asystole group (WHITE CIRCLE)
B
Death (BLACK CIRCLE) and
Short - Recovery Time
(<15min) (WHITE CIRCLE)
Asphyctic PRETERM newborns- University of Thessaloniki (Greece)
Prof. K. Sarafidis. Unsupervised analysis
(Prof. G. Buonocore (Siena) (Prof Fanos - Cagliari)
Mean spectra of
“Survival and
Control”
3 asphyctic TERM newborns
before and after
hypothermia (urinary
metabolomics)
Fanos V et al, Clin Biochem June, 2014
Courtesy of Prof. Manfred Spraul (Munich), 2014
Translational medicine: from top research to bedside (expected within 5 yeas)
FASEB J. 26,
2607–2619 (2012). www.fasebj.org
Metabolomica e Sepsi
(FUTURO IMMINENTE)
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Diagnosticare molto precocemente la sepsi
Diagnosticare l’eziologia
Diagnosticare la severità
Identificare chi risponde alla terapia
Identificare i soggetti a rischio di
progressione della malattia
Time sequence of markers during sepsis
(appearance of symptoms and signs)
IL-2
IL-6
0
PCT
SAA
IL-8
PCR
Presepsin
12h
24h
48h
Time sequence of markers during sepsis
(appearance of symptoms and signs)
Metabolomics (?)
IL-2
IL-6
0
PCT
SAA
IL-8
PCR
Presepsin
12h
24h
48h
On the subject of individual variability, we can conclude with a
sentence by Montaigne “there is a greater difference between
one man and another than there is between a man and an
animal”.
Only by being aware of complexity and biological variability,
by improving our knowledge, by feeding and treating different
individuals in different ways, and most of all by better
defining the state of health of each individual and his/her
resilience, will medicine be in a position to respond in a
personalized and customized way (and not approximately and
epidemiologically) to the problems of human health.
Acknowledgments
and permanent
collaborations
Luigi Atzori, Luigi Barberini
Scienze Neur. e Cardiovascolari,
University of Cagliari
Melania Puddu, Giovanni Ottonello,
Antonio Noto, NICU Cagliari
Emanuela Locci, Flaminia Marincola,
Paola Scano Dip. Scienze Chimiche,
University of Cagliari
Theodoros Xanthos
Medical School, National
University of Athens
Michele Mussap
San Martino Hospital, Univ. of Genoa
Giuseppe Buonocore, Univ. of Siena
Julian Griffin
Department of Biochemistry
University of Cambridge
Aalim Weljie
Metabolomics Research Centre
Univ. of Calgary
OUR INTERNATIONAL COLLABORATIONS ON METABOLOMICS
25 collaborations with 17 countries in 5 continents
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Australia, Brasil, Canada, USA, Mexico, Mozambico, Malaysia, Portugal, Sweden, Finland,
Ireland, Turkey, France, Greece, The Netherlands, United Kingdom, Switzerland
Metabolomics in sepsis: a step towards the future?
“As for the future,
your task is not to
foresee,
but to enable it”
Antoine de Saint-Exupéry
The Wisdom of the Sands
Proceedings
published on JPNIM
and Clin Chim Acta
3rd International Workshop on Neonatology,
Cagliari Ottobre 2006
Costituzione del Gruppo di Biochimica Neonatale
Prof. Giuseppe Buonocore (Siena)
Prof. Giovanni Corsello (Palermo)
Prof. Claudio Fabris (Torino)
Prof. Vassilios Fanos (Cagliari)
Dott. Diego Gazzolo (Alessandria)
Dott. Michele Mussap (Genova)
Dott. Paolo Tagliabue (Monza)
Dott. Rinaldo Zanini (Lecco)
BEST SPECIALISTS ON
BIOMARKERS and
METABOLOMICS
From:
•
•
•
•
•
•
Belgium
Israel
Germany
Switzerland
United Kingdom
Italy
Clinica Chimica Acta
Translational medicine:
from top research
to bedside
Concluding comments
• Metabolomics could rapidly become the “new clinical
chemistry”
• The first results are available in Neonatology
• Metabolomics could be a major application in sepsis
in the next future
• Personalized neonatal approach
International collaborations on Metabolomics
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Australia, Canada, USA, Mexico, Mozambico, Malaysia, Portugal,
Sweden, Turkey, France, Greece, Holland, United Kingdom, Switzerland
Metabolomics and BPD
Timing of urine collection
Birth
Day 7
Day 30
GA 38 wks
……………
Discharge
………
A work in progress
“Searching hub metabolites from birth”
Title: Aggression in Children: unraveling geneenvironment interplay to inform Treatment and
InterventiON strategies
FP7 Winner Project. Acronym: ACTION
METABOLOMICS
Partner
4
Partner
7
Partner
8
Partner
6
Partner
5
Partner
1Partner
Partner
2
Partner
11
12
Partner
10
Partner
9
Partner
3
Metabolomics and
perinatal
programming
Are the fetus and the
newborn father to the
man?
abstract
10
years
Congenital CMV infection:
Urine at birth predicts outcome
JPNIM-open access, 2012
(abstract)
7 years
Concluding comments
• Metabolomics could rapidly become the “new clinical
chemistry”
• The first results are available in Neonatology
• Nutri-metabolomics could be a major application in
the next future
• Importance in perinatal programming
• Personalized neonatal approach
Fanos V. et al. Metabolomics in Pediatric Nephrology. Molecules 2013, (mod)
Young adults (24 years) born ELBW (BW < 1 Kg)
Bassareo PP, Fanos V et al. Int J Cardiov Epidemiol 2012, Int J Cardiol
2011, JMFNM 2009, 2010, Acta Pediatrica 2010
Observed results
Practical consequences
Prolongation in QT interval
Risk of sudden death
Monitor ECG
Avoid drugs prolonging QT
Reduced brachial-flow mediated
vasodilatation
Risk of hypertension
Monitor blood pressure
High levels of asymmetric
dimethylarginine (ADMA), a
strong inhibitor of nitric oxide
synthesis
Risk of future cardiovascular
events and cardiac death
Monitor blood pressure,
echocardiography
Dramatic Increase of
microalbuminuria, uNGAL
(Neutrophil Gelatinase
Associated Lipocalin)
Decrease of renal volume
Risk of chronic renal failure
Monitor creatininemia, cystatin
C, microalbuminuria, urine
dipstick
Sotto l’Alto Patronato del
Presidente della Repubblica per
l’8° anno consecutivo
700 partecipanti circa 5
premi poster da 500 euro per
i giovani
Metabolomica e sepsi neonatale
Asphyxia =  
HISTOPATHOLOGY OF ASPHYXIA
IN NEWBORN PIGLETS
The animal
experiment was
performed at the
‘ELPEN’ Research
– Experimental
Centre in Athens,
Greece
MATERIAL AND METHODS
40 male Landrace / Large White newborn piglets
2 – 4 days old
2.5 ± 1.2 kg
randomly allocated in 4 groups according to the
concentration of O2 that was used for resuscitation
Group 1 →
Group 2 →
Group 3 →
Group 4 →
18% O2
21% O2 – control group
40% O2
100% O2
Resuscitation after hypoxia and reox at different oxygen concentration (in minutes) in
40 piglets
Oxygen concentration
number of death
100%
3
40%
2
1
21%
18%
3
0
10
Reox
Average in minute
Median in minute
standard deviation in
minute
20
30
40
50
60
70
80
90
100
minute
18%
21%
40%
100%
14.57
32.75
76.5
57
15
31
70
44
7.72
22.25
44.10
34.18
A metabolomic approach in an experimental model of hypoxiareoxygenation in newborn piglets: urine predicts outcome (JMFNM 2010)
Luigi Atzori, Theodoros Xanthos…., Nicoletta Iacovidou2
and Vassilios Fanos in collaboration with the University of Athens
A
Asystole group (BLACK CIRCLE)
Non-Asystole group (WHITE CIRCLE)
B
Death (BLACK CIRCLE) and
Short - Recovery Time
(<15min) (WHITE CIRCLE)
Pre and post-resuscitation values in piglets treated with
room air (FiO2: 21%)
Pig 1: death.
Glycine immodified pre
and post resuscitation
%
Pig 2: immediate
recovery after 7
minutes
FiO2
18%
FiO2
21%
FiO2
40%
FiO2
100%
Energy requirement
FiO2
18%
Homeostasis
FiO2
21%
Oxidative Stress
FiO2
40%
FiO2
100%
Oxidative Stress
Brain injury
Newborn piglet resuscitated with 100% oxygen: the brain shows
an extremely high number of apoptosis
(Collaboration with Prof. Gavino Faa (Cagliari)
What about kidney histology?
BLOOD
BRAIN
anoxia
ratios of alanine to
branched chained
amino acids
(Ala/BCAA;) and of
glycine to BCAA
(Gly/BCAA)
phosphocreatine, ATP
and ADP
Liu J JCBFBM 2011
URINE
urea
creatinine
malonate
metilghuanidine
uric acid
hypoxanthine
malonylaldeide
Banupryia C Clin
Bioch 2008.
Atzori L JMFNM 2010
Reoxygenation
alpha keto-glutarate,
succinate and
fumarate
Solberg R PLoS One
2010
Fanos V et al.
JMFNM 2012
EURAIBI
Metabolomics and exp.
asphyxia
Pediatric Piglets