Biopatologia 2

Licenciatura em Ciências Bimédicas

2019-2020

Metabolomics Applied to Health

Sciences

Alexandra Nunes|alexandranunes@ua.pt
AN | 2020 1
Outline

¤  Defini&ons  

¤  History  

¤  Applica&ons  

¤  A  metabolomic  study  design  

¤  Metabolomic  tools  

¤  Applica&on  in  cancer  diagnosis  

¤  Concluding  remarks  

AN | 2020 2
Metebolomics includes the analysis and
interpretation of a large volume of data and
is useful tool to study health and disease.

AN | 2020 3
Definitions

AN | 2020 4
•  Metabolomics  
–  Newly  emerging  field  of  'omics'  research    
–  Comprehensive  and  systema&c  determina&on  of  metabolite  levels    in  the  
metabolome    and  their  changes  over  &me    
•  Metabolome  
–  Refers  to  the  complete  set  of  small-­‐molecule  metabolites  
–  Is  dynamic    
•  Metabolites  
–  Intermediates  and  products  of  metabolism  
–  Examples  include  an&bio&cs,  pigments,  carbohydrates,  faGy  acids  and  amino  acids  

AN | 2020 5
•  Genomics  is  the  study  of  DNA  and  gene&c  informa&on  within  a  cell,  and  
transcriptomics  is  the  study  of  RNA  and  differences  in  mRNA  expression;  
metabolomics  is  the  study  of  substrates  and  products  of  metabolism,  which  
are  influenced  by  both  gene7c  and  environmental  factors.  

AN | 2020 6
•  Large-­‐scale   study   of   small   molecules,   commonly   known   as   metabolites,  
within  cells,  biofluids,  &ssues  or  organisms.  

•  Collec&vely,  these  small  molecules  and  their  interac&ons  within  a  biological  

system  are  known  as  the  metabolome.  

An  overview  of  the  four  major  "omics"  fields,  from  genomics  to  
metabolomics.  

AN | 2020 7
=  high-­‐throughput  analysis  of  metabolites  
•  because  it  includes  the  measurement  of  of  a  large  number  of  cellular  
metabolites  (several  hundred).  

•  many  of  the  metabolites  found  are  not  iden&fied  and  the  result  of  the  
analysis  (i.e.  depending  on  the  metabolomic  approach  used  the  result  is  a  
profile).  

AN | 2020 8
A  small  molecule  (or  metabolite)  is  a  low  molecular  weight  organic  compound,  typically  
involved  in  a  biological  process  as  a  substrate  or  product.    
Metabolomics  usually  studies  small  molecules  within  a  mass  range  of  50  –  1500  daltons  
(Da).  
 
Some  examples  of  small  molecules  include:  sugars,  lipids,  amino  acids,  faGy  acids,  phenolic  
compounds...  

AN | 2020 9
A  small  molecule  (or  metabolite)  is  a  low  molecular  weight  organic  compound,  typically  
involved  in  a  biological  process  as  a  substrate  or  product.    
Metabolomics  usually  studies  small  molecules  within  a  mass  range  of  50  –  1500  daltons  
(Da).  
 
Some  examples  of  small  molecules  include:  sugars,  lipids,  amino  acids,  faGy  acids,  phenolic  
compounds...  

AN | 2020 10
AN | 2020 11
 1  GENE      →        1  mRNA      →        1  Protein  →    Many  Metabolites    
(and  conversely:            different    proteins    →  1  Metabolite)  
 
There  is  no  direct  rela&onship  between  metabolite  and  gene  in  the  way  there  is  
between  genes  and  mRNAs  and  proteins.      
 
A  single  gene  does  not  specify  the  level  of  a  single  metabolite,  although  it  may  
determine  whether  the  metabolite  is  present  or  absent.    

The  level  of  a  metabolite  is  determined  by  the  ac&vi&es  of  all  the  enzymes  of  all  
the  pathways  that  involve  that  metabolite,  and  by  effectors  that  act  on  these  
enzymes.      
 
Metabolite  levels  change  according  to  developmental,  physiological,  and  
pathological  states.  

AN | 2020 12
Many  reac&ons  take  place  con&nuously  within  cells,  so  concentra&ons  of  metabolites  
are  considered  to  be  very  dynamic,  and  may  change  rapidly  from  one  &me  point  to  the  
next.    
 
Current  analy&cal  techniques  used  to  inves&gate  metabolomics  can  only  take  a  snapshot  
in  &me  under  a  set  of  defined  condi&ons.  

Metabolic  pathways  are  essen&ally  a  series  of  chemical  reac&ons,  catalyzed  by  enzymes,  
where  the  product  of  one  reac&on  becomes  the  substrate  for  the  next  reac&on.  

AN | 2020 13
Includes  the  metabolic  and  physical  processes  
that  determine  the  physiological  and  
biochemical  proper&es  of  an  organism  
(includes  molecules,  chemical  reac&ons  of  
metabolism,  the  metabolic  pathways,  and  the  
regulatory  interac&ons).  
 
Metabolic  networks  can  be  used  to  detect  
comorbidity  paGerns  in  diseased  pa&ents.  
 
The  disease  phenotype  are  usually  the  
consequence  of  the  cell  to  breakdown  or  
produce  an  essen&al  substrate  and  o[en  only  
one    enzyme  defect  of  a  par&cular  reac&on  
affect  other  subsequent  reac&ons.  These  
chain  effects  associate  to  a  metabolic  disease  
several  comorbidity  effects.  The  metabolic  
disease  networks  can  be  used  to  determine  if   Where  to  look  for  metabolic  network:  
two  disorders  are  connected  due  to  their   Kyoto  Encyclopedia  of  Genes  and  Genomes  (KEGG),  EcoCyc,BioCyc  

correlated  reac&ons.  
AN | 2020 14
The  metabolome  is  
dependent    of  the  metabolic  
pathways  and  can  be  altered  
by  changes  in  each  one.  

AN | 2020 15
History

AN | 2020 16
•  Metabolic  profiling  is  not  new.    Profiling  for  clinical  detec&on  of  human  disease  using  
blood  and  urine  samples  has  been  carried  out  for  Centuries.  

This  urine  wheel  was  

published  in  1506  by  
Ullrich  Pinder,  in  his  book  
Epiphanie  Medicorum.    
 
The  wheel  describes  the  
possible  colors,  smells  
and  tastes  of  urine,  and  
uses  them  to  diagnose  
disease.    

Nicholson,  J.  K.  &  Lindon,  J.  C.  Nature  

455,  1054–1056  (2008).  
AN | 2020 17
•  The  first  paper  that  reported  a  study  of  metabolome  
–  “Quan&ta&ve  Analysis  of  Urine  Vapor  and  Breath  by  Gas-­‐Liquid  Par&&on  
Chromatography”,  by  Robinson  and  Pauling  in  1971.    
Abstract  
When  a  human  being  is  placed  for  several  days  on  a  completely  defined  diet,  
consis;ng  almost  en;rely  of  small  molecules  that  are  absorbed  from  the  
stomach  into  the  blood,  intes;nal  flora  disappear  because  of  lack  of  nutri;on.  
By  this  technique,  the  composi;on  of  body  fluids  can  be  made  constant  
(standard  devia;on  about  10%)  aFer  a  few  days,  permiHng  significant  
quan;ta;ve  analyses  to  be  performed.  A  method  of  temperature-­‐programmed  
gas-­‐liquid  par;;on  chromatography  has  been  developed  for  this  purpose.  It  
permits  the  quan;ta;ve  determina;on  of  about  250  substances  in  a  sample  of  
breath,  and  of  about  280  substances  in  a  sample  of  urine  vapor.  The  technique  
should  be  useful  in  the  applica;on  of  the  principles  of  orthomolecular  
medicine.  

AN | 2020 18
•  The  name  metabolomics  was  used  in  the  late  1990s    
–  Oliver,  S.  G.,  Winson,  M.  K.,  Kell,  D.  B.  &  Baganz,  F.  (1998).  Systema&c  
func&onal  analysis  of  the  yeast  genome.    
Abstract  
The  genome  sequence  of  the  yeast  Saccharomyces  cerevisiae  has  provided  the  
first  complete  inventory  of  the  working  parts  of  a  eukaryo;c  cell.  The  
challenge  is  now  to  discover  what  each  of  the  gene  products  does  and  how  
they  interact  in  a  living  yeast  cell.  Systema;c  and  comprehensive  approaches  
to  the  elucida;on  of  yeast  gene  func;on  are  discussed  and  the  prospects  for  
the  func;onal  genomics  of  eukaryo;c  organisms  evaluated.  
 
“The  metabolome”  was  the  &tle  given  to  ne  of  the  subsec&on  of  the  paper.  

Many  of  the  bioanaly&cal  methods  used  for  metabolomics  have  been  adapted  
(or  adopted)  from  exis&ng  biochemical  techniques  and  were  used  in  other  
type  of  samples.  

AN | 2020 19
•  Since  than  the  publica&on  on  metabolomics  area  are  dras&cally  increasing:  

AN | 2020 20
Tools  that  were  built  with  metabolomics  to  metabolomics  

Human  Metabolome  project  –  first  dra[  of  human  metabolome  in  2007  
hGp://www.hmdb.ca  
The  Human  Metabolome  Database  (HMDB)  is  a  freely  available  electronic  
database  containing  detailed  informa&on  about  small  molecule  metabolites  
found  in  the  human  body.  It  can  be  used  for  in  metabolomics,  clinical  
chemistry,  biomarker  discovery  and  general  educa&on.  The  database  is  
designed  to  contain  or  link  three  kinds  of  data:  1)  chemical  data,  2)  clinical  
data,  and  3)  molecular  biology/biochemistry  data.  The  database  contains  
114,186  metabolite  entries.  

AN | 2020 21
Tools  that  were  built  with  metabolomics  to  metabolomics  

Small  Molecule  Pathway  Database  

hGp://smpdb.ca    
SMPDB  (The  Small  Molecule  Pathway  Database)  is  an  interac&ve,  visual  
database  containing  more  than  30  000  small  molecule  pathways  found  in  
humans  only.  The  majority  of  these  pathways  are  not  found  in  any  other  
pathway  database.  SMPDB  is  designed  specifically  to  support  pathway  
elucida&on  and  pathway  discovery  in  metabolomics,  transcriptomics,  
proteomics  and  systems  biology.    

AN | 2020 22
Applications

AN | 2020 23
 Metabolome

•  The  metabolome  is  the  total  complement  of  metabolites  present  in  a  biological  
sample  under  given  gene&c,  nutri&onal  or  environmental  condi&ons  –  is  
dimamic.  
 
•  Metabolomics  technologies  yield  many  insights  into  basic  biological  research  in  
areas  such  as  systems  biology  and  metabolic  modelling,  pharmaceu&cal  
research,  nutri&on  and  toxicology.  
AN | 2020 24
Unlike  other  "omics"  measures,  metabolites  and  their  concentra&ons,  reflect  
directly  the  underlying  biochemical  ac&vity  and  state  of  cells  /  &ssues.    
 
Thus  metabolomics  best  represents  the  molecular  phenotype.  

AN | 2020 25
  
•  non-­‐invasive  nature  (prevalence  has  been  done  to  non-­‐invasive  sampling,  
using  biological  fluids  such  as  saliva,  urine,  blood  (serum  or  plasma)  but  
&ssue  are  also  possible  to  test  (biopsies)  or  more  invasive  samples  (CSF)  can  
also  be  used  

•  close  link  to  the  phenotype  (give  insight  of  the  func&onality  of  all  metabolic  
pathways)  
 
These  characteris&cs  make  metabolomics  an  ideal  tool  for  the  pharmaceu&cal  
(and  drug  safety  screens),  preven&ve  healthcare  (biomarker  discovery  )  and  
personalized  medicine  (it  will  be  possible  to  use/develop  personalized  drugs  and  
improved  treatment  strategies).    
 
Personalized  treatment  is  likely  to  be  more  effec7ve  than  current  medical  
popula7on-­‐based  approaches.  

AN | 2020 26
Benefit  from  metabolomics  on  various  levels:  medical  diagnos&cs  in  
healthcare,  and  future  applica&ons  in  personalized  medicine  resul&ng  in  
personalized  treatment  strategies.  

•  Forensics  
•  How  did  he  die?        
•  Response  to  environmental  changes            
•  Molecular  diagnosis    
•  Pathway  discovery    
•  “Systems  Biology”  
•  Integrated  knowledge  
•  Personalized  medicine  
•  One  to  one,  drug  discovery,  drug  design  
•  In  deep  pathology  study    
•  Predic&ve  medicine  

AN | 2020 27
A[er  accurate  model  development,  metabolomics'  based  diagnosis  enables  a  real  &me  
results.  

AN | 2020
Biomarker  discovery  is  an  area  where  metabolomics  informs  decision  making.  
Biomarkers  are  "objec&ve  indica&ons  of  medical  state  observed  from  outside  
the  pa&ent  -­‐  which  can  be  measured  accurately  and  reproducibly"  (Kyle  et  al.  
What  are  Biomarkers?).  
 
In  metabolomics,  biomarkers  are  small  molecules  (metabolites)  that  can  be  
used  to  dis7nguish  two  groups  of  samples,  typically  a  disease  and  control  
group.    
 
•  A  metabolite  present  in  disease  samples,  but  not  in  healthy  individuals  
would  be  classed  as  a  biomarker.    
•  Samples  of  urine,  saliva,  bile,  or  seminal  fluid  contain  highly  
informa&ve  metabolites,  and  can  be  analyzed  through  metabolomics  
fingerprin&ng  or  profiling,  for  the  purpose  of  biomarker  discovery.  

AN | 2020 29
Personalized   medicine,   the  
u l & m a t e   c u s t o m i z a & o n   o f  
h e a l t h c a r e ,   r e q u i r e s  
metabolomics   for   quick   medical  
diagnosis  to  iden&fy  disease.    
•  I n   h e a l t h c a r e ,   c l a s s i c a l  
biochemical   tests   are   used   to  
measure   individual   metabolite  
concentra&ons   to   iden&fy  
disease   states   (e.g.   the   blood-­‐
glucose  level  in  diabetes).  
•  M e t a b o l o m i c s   o ff e r s   t h e  
p o t e n & a l   f o r   t h e   r a p i d  
iden&fica&on   of   hundreds   of  
metabolites,   enabling   us   to  
iden&fy   these   disease   states  
much  earlier.  
AN | 2020 30
Examples  of  devices  form  biomarker  diagnosis  and  monitoring      

AN | 2020 31
Varia&ons  of  molecules  concentra&on  in  the  organism  and  how  the  varia&ons  can  be  
used  in  different  contexts:  
Metabolites  levels  

AN | 2020 32
Personalized  medicine,  precision  medicine,  P4  medicine  (P4=predic&ve,  preven&ve,  personalized  
and  par&cipatory)  and  systems  medicine  are  different  names  to  illustrate  the  common  desire  to  
establish  a  novel  (more  personalized,  precise  and  systema&c)  approach  in  medicine.    
Systems  medicine  as  an  integra&ve  approach,  combining  technologies,  data,  methodologies  and  
exper&se.    

Experimental  &  Molecular  Medicine  (2018)  50,  e453;  doi:10.1038/emm.2017.290  

AN | 2020 33
 Outline  of  the  systems  medicine  
ra&onale.  The  transforma&on  of  
diverse  prior  knowledge  and  
newly  generated  data  into  
hypotheses  using  computa&onal  
and  mathema&cal  methods,  
tools.  

Systems  Biology  and  Applica&ons  (2018)4:21;  doi:10.1038/s41540-­‐018-­‐0059-­‐y    

  AN | 2020 34
AN | 2020 35
The  two  main  approaches  that  can  be  used  in  metabolomics  are  untargeted  and  
targeted  approaches.  

measures  as  many  metabolites  as  possible  from  a  range  of  biological  
samples  without  any  (intended)  bias.  

is  used  when  you  want  to  measure  sets  of  metabolites  and  have  a  specific  
biochemical  ques&on  that  you  want  to  answer.  
This  approach  is  o[en  used  in  pharmacokine&c  studies  of  drug  metabolism  
and  when  looking  at  the  effect  of  therapeu&cs  or  gene&c  modifica&ons  on  a  
specific  enzyme.  

AN | 2020 36
Example  of  an  untargeted  and  a  targeted  approach.  

AN | 2020 37
Pipeline  for  experimental  design  based  in  metabolomics.  

AN | 2020 38
Sampling MS;  NMR;  FTIR Data Data  Analysis Interpreta&on

AN | 2020 39
 •  What  will  be  the  
correct  data  set  (n)?  
What  do  you  want  to  study?  
•  How  will  be  the  
How  are  you  going  to  study?   control  samples?  
•  How  will  the  
volunteers  will  be  
Important  thoughts  when  design  a  metabolomics  study  
gathered?  
•  How  to  obtain  an  
ethics  approval?  
•  Which  
metabolomic  
technique  will  
be  used?  
•  Which  tools  
will  extract  the  
informa&on  
needed?  

•  What  metabolite  will  be  studied?  

•  It  will  be  needed  a  extrac&on/
concentra&on  of  the  metabolite?  
•  Is  it  possible  to  analyze  directly  the  
sample  without  pre-­‐treatment?   AN | 2020 40
Sample  prepara&on  steps:  collec&on,  storage,  extrac&on,  prepara&on,  custom  prepara&on  
 for  individual  measurement  systems  (eg  deriva&za&on  for  gas  chromatography).  
Note:  No-­‐sample  prepara&on  is  also  possible  (eg.  FTIR).    

AN | 2020 41
Metabolites  are  typically  extracted  in  aqueous  or  methanolic  media,  then  frac&onated  
into  lipophilic  and  polar  phases  that  are  then  analyzed  separately.    
The  frac&ona&on  of  each  phase  may  follow  to  split  metabolites  into  classes  prior  to  
analysis.  

No  single  extrac7on  procedure  works  for  all  metabolites  because  condi&ons  that  
stabilize  one  type  of  compound  will  destroy  other  types  or  interfere  with  their  analysis.  
Therefore  the  extrac7on  protocol  has  to  be  tailored  to  the  metabolites  to  be  profiled.  

AN | 2020 42  
Metabolic  profiling  is  o[en  confined  to  fairly  stable  compounds  that  can  be  extracted  
together.      
 
The  most  comprehensive  profiling  can  cover  several  hundred  compounds,  many  of  which  
are  uniden&fied.      
Many  crucial  metabolites,  par&cularly  minor  or  unstable  ones,  are  currently  being  missed  
in  metabolomics  analyses.    
 
Methodologies  for  the  analysis  of  less  stable  ones  are  needed.    
Non-­‐destruc7ve  methodologies  that  do  not  require  pre-­‐treatment  are  necessary.  
 
Due  to  the  complexity  of  the  metabolome  and  the  diverse  proper7es  of  
metabolites,  no  single  analy7cal  plaOorm  can  be  applied  to  detect  all  
metabolites  in  a  biological  sample.  

AN | 2020 43
 Selected biological sample

metabolites  

proteins RNA

Weckwerth,  2003  “Metabolomics  in  Systems  Biology”  

AN | 2020 44
 disease  group  
Popula&on  
 ê  
Disease  

Outcome:   control  group  

Metabolomic  
•  Scien&fic  knowledge   Data  Acquisi7on    
Methodology  of  
•  Biomarkers  iden&fica&on  
Analysis  
•  Diagnosis  and  prognosis  methods  
•  Development  of  new  drugs  
•  Personalized  medicine  
•  Predicted  medicine  
sample  collec&on  
Data  Analysis  
sample  prepara&on  
(qualita7ve  or  
clinical  informa&on  
quan7ta7ve)  

•  External  valida&on  
•  Classifica&on  model  

•  Metabolite  iden&fica&on  
•  Metabolite  profile  iden&fica&on  
•  Pathway  analysis  

AN | 2020 45
Metabolomic tools

AN | 2020 46
NMR

FTIR

MS

AN | 2020 47
MS  (Mass  Spectrometry)  is    combined  with  chromatography  [LC  or  GC]    
•  Most  widely  used,  par&cularly  produc&ve  for  LMW  compounds  (pep&des  as  
well).  
•  Needs  a  previous  separa&on  by  a  chromatographic  technique  (Liquid  –  LC  or  
Gas  –  GC).  
•  In  GC/MS  the  sample  must  become  vola&le,  which  requires  deriva&za&on.    
•  In  LC/MS,  without  deriva&za&on,  compound  groups  must  be  
“selected”  (size,  chemical  proper&es)  by  the  choice  of  columns  or  isola&on  
procedures.    
•  A[er  separa&on  the  compound  are  ionized  and  separated  and  detected  
according  to  mass  tocharge  ra&o  (m/Z).

AN | 2020 48
 Modules  of  a  simple  Mass  Spectrometer.  

•  To  iden&fy  and  to  quan&fy  

metabolites  
•  Serves  to  both  separate  and  to  
detect  mass  to  charge  ra&os  
•  Rela&vely  low  cost    
•  High  separa&on  efficiency    
•  Separa&on  of  several  hundred  
compounds  per  run  

AN | 2020 49
•     GC/MS  –  Gas  Chromatography  +  Mass  Spectrometry    

Compounds  must  be  deriva&zed  to  become  vola&le  

AN | 2020 50
•     LC/MS  –  Liquid  Chromatography  +  Mass  Spectrometry  
•  Separa&on  even  beGer  than  GC/MS    
•  No  deriva&za&on  necessary    
•  LC/MS  is  more  suitable  than  GC/MS  for  labile  compounds,  for  those  that  
 are  hard  to  deriva&ze,  or  hard  to  render  vola&le.      

AN | 2020 51
Overlapping  peaks  can  be  deconvoluted  because  the  spectra  of  their  cons&tuents  
are  dis&nct  

Target  metabolites  are  iden&fied  by  exact  reten&on  &mes  and  their  corresponding  mass  
spectra.  Huge  number  of  chemical  structures  can  have  the  same  exact  mass!  
AN | 2020 52
Advantages   Disadvantages  
•  It  is  much  more  sensi&ve  than  NMR.   •  Similarity  of  isomers  can  make  
This  mean  that  can  study  more   iden&fica&on  difficult.  
metabolites.     •  For  LC-­‐MS,  libraries  and  not  complete.  
•  30-­‐100  metabolites  can  be  iden&fied   •  For  GC-­‐MS  samples  must  be  
from  NMR  data  whilst  GC-­‐MS  allowed   deriva&sed  before  analysis  which  
the  iden&fica&on  of  >300  metabolites   increases  the  prepara&on  &me.    
in  the  aqueous  and  >  1000  for  LC-­‐MS  
•  Not  limited  to  samples  type  and  
poten&ally  more  global    
•  Takes  up  much  less  space  than  an  
NMR  machine  and  cheaper  to  buy  
•  Less  expensive  equipment  than  NMR    

AN | 2020 53
•  NMR  (Nuclear  Magne&c  Resonance)  
Allows  a  metabolite  fingerprints  for  compounds  with  non-­‐zero  magne&c  
moments;  atoms  with  uneven  number  of  electrons  (best:  1H,  13C,  19F,  31P).    
Samples  can  be  analyzed  without  pretreatment.  
It  is  a  good  op&on  for  metabolic  screening.  

AN | 2020 54
•  A  current  generates  a  strong  magne&c  
field  and  polarizes  the  nuclei  in  the  
sample  material  -­‐  radio-­‐frequency  (RF)    
•  Spectra  shows  "lines"  for  different  
nuclei  in  different  electronic  
environments.  
•  Many  atoms  have  nuclei  that  are  NMR  
ac&ve,  but  most  NMR  data  are  
collected  for  1H  and  13C  .  
•  The  main  weakness  of  NMR  is  low  
sensi&vity  rela&ve  to  MS.  It  is  less  
suited  for  analysis  of  trace  compounds  
•  Does  not  destroy  the  sample;  can  
detect  and  quan&fy  metabolite  
because  the  signal  intensity  is  only  
determined  by  the  molar  
concentra&on;  can  provide  
comprehensive  structural  informa&on.  

AN | 2020 55
The  informa&on  obtained  is  displayed  as  a  spectrum.    The  horizontal  axis  is  the  
chemical  shi[  (delta,  in  units  of  ppm),  which  is  a  measure  of  the  posi&on  at  which  RF  
absorp&on  occurs  rela&ve  to  an  internal  standard  (tetramethylsilane,  TMS).    
The  ver&cal  axis  is  the  intensity  of  the  absorp&on.    As  with  other  spectral  techniques,  
compounds  have  characteris&c  spectra.  

A typical 950-MHz H NMR spectrum of urine showing the degree of spectral complexity
AN | 2020 56
Advantages   Disadvantages  
•  NMR  does  not  rely  on  separa&on  of   •  It  is  not  as  sensi&ve  as  MS  techniques.    
the  analytes.     •  As  a  consequence  high-­‐  resolu&on  
•  The  sample  is  not  destroyed  during   NMR  of  intact  biofluids  does  not  yet  
analysis.     iden&fy  all  the  metabolites.    
•  All  kinds  of  small  molecule  metabolite   •  Expansive  equipment.  
can  be  measured  simultaneously.    
•  Provides  good  fingerprint  of  most  
metabolites.    
•  Sample  analysis  is  fast  (~7  minutes)  
and  robust,  enabling  high  throughput  
(if  sample  is  liquid).    

AN | 2020 57
FT-­‐IR  (Fourier-­‐transform  Infrared  Spectroscopy)    
IR-­‐radia&on  interacts  with  compounds  and  is  recorded  its  absorp&on  and  its  
intensity.    
A  spectrum  is  obtained  that  measures  the  vibra&ons  of  chemical  bond  of  
func&onal  groups.  
 The  peak  posi&on  of  the  spectra  indicates  the  func&onal  groups  present  and  
consequently  the  molecules  present  in  the  sample.    
The  spectrum  peak  posi&on  is  compared  with  literature.    

AN | 2020 58
FT-­‐IR  is  a  type  of  absorp&on  spectroscopy  that  uses  the  infrared  region  of  the  
electromagne&c  spectrum.  
 
Useful  tool  for  iden&fying  covalent  molecular  bonds  present  in  the  irradiated  
molecule,  since  it  gives  rise  to  an  absorp&on  spectrum  which  is  a  molecular  
fingerprint.  
 
The  IR  radia&on  when  absorbed  provides  enough  energy  only  to  change  the  
vibra&ons  between  the  atoms  in  a  molecule.  

AN | 2020 59
Advantages   Disadvantages  
•  Sample  can  be  analysed  directly   •  It  is  not  as  sensi&ve  as  NMR  or  MS  
without  pre-­‐treatment  nor  internal   techniques.    
standard.  The  sample  is  not  destroyed   •  It  is  not  consensually  a  metabolomics  
and  can  be  used  in  other  experiments.     technique  because  it  doesn't  give  the  
•  All  kinds  of  small  molecule  metabolite   exact  composi&on  of  the  sample,  it  is  
can  be  measured  simultaneously  –  it  is   possible  to  iden&fy  the  family  of  the  
possible  to  obtain  an  overall   molecules  present  in  the  sample.    
composi&on  of  the  sample.    
•  Sample  analysis  is  fast,  ~1  minute  per  
replicate  –  good  technique  for  
screening.  
•  Equipment  has  a  very  low  price.    

AN | 2020 60
Application in cancer diagnosis

AN | 2020 61
 Metabolomics
Spectroscopic Technique and
Multivariate analysis

Healthy Aging Dementia Cancer

AN | 2020 62
Data set
!

Number!of!patients! Number!of!prostate!samples! Benign! Malignant!

8! 16! 8! 8!
!

Experimental design

AN | 2020 63
Results – FTIR spectra

Difficult  to  observe  

Main  differences  
differences  between  
Main  differences   spectra  of  NPT  and  PTT  
samples.    
To  visualize  the  differences  
between  spectra  and  
therefore  between  
samples  mul&variate  
analysis  are  used.    
 
This  sta&s&c  tools  allow  to  
discriminate  the  samples  
and  explain  why  the  
samples  are  different.      

AN | 2020 64
 Results – Multivariate analysis (Principal Component Analysis)
Histological  “normal”  control  with  metabolomic  altera&ons  
consistent  to  tumoral  profile.   C - control (NPT)
T – tumoral (PTT)
Each dot – 1 spectra
more  aggressive  tumor  
A discrimination between
NPT and PTT is observed.
tumoral
Sample 5 correspond to
the more aggressive
prostate tumor.
Sample 8 correspond to
control benign prostatic
hyperplasia.

Most relevant metabolic

tumoral alterations observed:
§  Deregulation in lipid
metabolism,
§  Decrease in
polysaccharide and
glycogen content,
control §  Increase in nucleic
acids content and
benign  prosta&c  hyperplasia  sample   protein
phosphorylation
AN | 2020 65
Concluding remarks
Usually  a[er  collec&ng  the  &ssue  the  histopathologist  analyzes  it  under  an  op&cal  
microscope  trying  to  answer  a  few  ques&ons:  Is  the  &ssue  normal?  Is  it  a  benign  change  
or  cancer?  How  aggressive  is  the  tumor?  How  likely  is  it  to  target  other  organs?  
FTIR  can  help  to  answer  these  ques&ons  in  the  following  ways  objec&ve,  quick,  non-­‐
destruc&ve  (&ssue  is  preserved)  and  requiring  a  small  amount  of  sample  without  any  pre-­‐
treatment.  
 
In  fact  biopsy  &ssue  can  be  analyzed  directly  by  FTIR  and  FTIR  can  be  used  as  a  method  of  
diagnose  and  classify  tumors.  
 
FTIR  provides  a  biochemical  signature.  Correlates  morphology  with  the  molecular  biology  
of  discrete  regions  of  the  tumor.  
Helps  the  diagnosis  (classifica&on  of  &ssue  and  assessment  of  aggressiveness  or  tumor  
stage)  reducing  subjec&vity.    

AN | 2020 66
Concluding remarks

¤  Metabolomics  is  the  large-­‐scale  study  of  small  molecules  (metabolites)  
within  cells,  &ssues  or  organisms.  

¤  Applica&ons  of  metabolomics  are  found  within  the  pharmaceu&cal,  

forensic,  diagnos&c  and  prognos&c  areas,  among  others.  

¤  There  are  two  main  approaches  used  in  metabolomic  studies:  untargeted  
(global)  and  targeted  (specific).  

¤  Careful  planning  and  design  of  experiments  is  of  paramount  importance  in  
metabolomic  studies.  

¤  NMR,  MS  and  FTIR  are  the  most  commonly  used  analy&cal  methods  in  
metabolomic  studies.  

AN | 2020 67