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Department of Chemistry

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Collection / analysis of more samples. 2. Validation of results by chemometrics ... Qu mica, UA, Aveiro. Dr. Eberhard Humpfer Bruker BioSpin GmbH ... – PowerPoint PPT presentation

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Title: Department of Chemistry


1
Assessing the Metabolic Phenotype of Cancer by
Nuclear Magnetic Resonance (NMR)
Iola F. Duarte
Department of Chemistry University of Aveiro
4th July 2008 Encontro com os Laboratórios
Associados
2
I. Introduction NMR in Biomedical Research
2
Why using NMR?
  • Direct analysis of complex biological
    samples
  • Non-selective and simultaneous detection of
    endogenous metabolites
  • Quantitative (min. 10 µM)
  • Rapid (1D 10 min)
  • Non-destructive

3
I. Introduction NMR in Biomedical Research
2
Why using NMR?
500 MHz 1H NMR spectra
Urine
  • Direct analysis of complex biological
    samples
  • Non-selective and simultaneous detection of
    endogenous metabolites
  • Quantitative (min. 10 µM)
  • Rapid (1D 10 min)
  • Non-destructive

Lung Biopsy
Endometrial cells
4
I. Introduction NMR in Biomedical Research
3
From spectral to biochemical information
Metabolic profile (identification /
quantification)
1D 2D LC-NMR/(MS) etc.
Physiological status
5
I. Introduction NMR in Biomedical Research
3
From spectral to biochemical information
Physiological status
6
I. Introduction NMR in Biomedical Research
4
Tools NMR, LC-MS Chemometrics
7
I. Introduction NMR in Biomedical Research
5
Potential cancer biomarkers found by
NMR-metabonomics
8
II. Results NMR of Lung Tumors
6
9
II. Results NMR of Lung Tumors
6
10
II. Results NMR of Lung Tumors
6
PTDC/QUI/68017/2006 (Jan 2008 Jan 2011)
Metabolic Profiling and Biochemical
Differentiation of Human Lung Tumors by NMR
Methods
Biofluids
Tissues
Cells
Hosting Inst. Univ. Aveiro (PI I.F.Duarte A.M.
Gil, A. Barros, B.J. Goodfellow) Participants
Hospitais Univ. Coimbra (L. Cavalho, V. Sousa, J.
Bernardo, L.E. Fernandes) Fac. Medicina Univ.
Coimbra (I.M. Carreira, J. Melo)
11
II. Results NMR of Lung Tumors
7
Research Questions
1. What does NMR tell us about human lung
composition?
2. How different are tumoral and normal adjacent
tissues?
3. How different are lung cancer histological
types?
4. Is there a metabolic phenotype of lung cancer
or its histological types?
5. How can this phenotype be used in diagnosis,
prognosis or therapeutic planning?
12
II. Results NMR of Lung Tumors
8
What can NMR tell us about human lung composition?
500 MHz 1H HRMAS-NMR Spectra (High Resolution
Magic Angle Spinning)
13
II. Results NMR of Lung Tumors
9
What can NMR tell us about human lung composition?
500 MHz 2D HRMAS-NMR spectra
TOCSY (1H-1H)
HSQC (1H-13C)
13C
1H
14
II. Results NMR of Lung Tumors
10
What can NMR tell us about human lung composition?
Compounds Identified 21 aminoacids ile, leu,
val, thr, ala, b-ala, arg, lys, met, glu, gln,
asn, asp, gly, pro, phe, tyr, creatine,
creatinine, taurine, carnitine 3 organic acids
lactate, formate, fumarate 3 alcohols glycerol,
myo-inositol, scylo-inositol 2 aminoalcohols
ethanolamine, phosphoethanolamine 3 choline
metabolites choline, phosphocholine,
glycerophosphocholine 4 bases and derivatives
inosine, uracil, hypoxanthine, uridine Phospholip
ids (phosphatidylcholines) oligopeptides
15
II. Results NMR of Lung Tumors
11
How different are tumoral and normal adjacent
tissues?
16
II. Results NMR of Lung Tumors
12
Energy Production in Cells
Warburg Effect
17
II. Results NMR of Lung Tumors
13
How different are tumoral and normal adjacent
tissues?
1H HRMAS-NMR (600 MHz)
4.3 2.9 ppm
Normal Tumoral epidermoid carcinoma
gt Lactate
18
II. Results NMR of Lung Tumors
14
How different are tumoral and normal adjacent
tissues?
1H HRMAS-NMR (600 MHz)
9.0 5.6 ppm
Normal Tumoral epidermoid carcinoma
19
II. Results NMR of Lung Tumors
15
First steps in the metabolic phenotype of lung
cancer
20
II. Results NMR of Lung Tumors
16
Biochemical information retrieved from spectral
variations
  • Increased glycolytic activity glucose
    conversion to lactate and variations in
    several amino acids
  • Increased gluconeogenesis degradation of
    triglycerides to meet the cells higher
    energetic needs
  • Accelerated cell proliferation variations in
    phospholipids, choline and
    inositol

21
II. Results NMR of Lung Tumors
17
Further steps
1. Collection / analysis of more samples
22
Acknowledgements
Prof.ª Lina Carvalho Inst. Anat.
Patológica, HUC/FMUC, Coimbra Dr. Vitor Sousa
Inst. Anat. Patológica, HUC/FMUC,
Coimbra Dr. João Bernardo C.
Cirurgia Cardiotorácica, HUC, Coimbra Prof. Luís
Eugénio C. Cirurgia
Cardiotorácica, HUC, Coimbra Prof.ª Isabel M.
Carreira Lab. Citogenética, FMUC,
Coimbra Dra. Joana Melo Lab.
Citogenética, FMUC, Coimbra Prof.ª Ana M. Gil
CICECO, Dep. Química,
UA, Aveiro Prof. Brian J. Goodfellow
CICECO, Dep. Química, UA, Aveiro Dr. Eberhard
Humpfer
Bruker BioSpin GmbH Dr. Manfred Spraul
Bruker BioSpin
GmbH
23
Thank you!
24
I. Introduction NMR in Oncological Research
11
25
II. HRMAS-NMR of Lung Tumor Tissues
21
What can NMR tell us about human lung composition?
500 MHz 1H HRMAS-NMR
T2 Edited (CPMG)
2.9 to 0.7 ppm
Ala
Lactate Thr
Ile, Leu, Val
GluGln
Met
Glu
Glu
Pro
b-Ala
ArgLys
Gln
Asn/Asp
26
II. HRMAS-NMR of Lung Tumor Tissues
22
What can NMR tell us about human lung composition?
500 MHz 1H HRMAS-NMR
2.9 to 0.7 ppm
Diffusion Edited
FA fatty acyl chains of glycerides and
phospholipids
FA CH2-CH2-CO
FA CH2-CH2-CHCH N-Ac oligopeptides
FA CH2-CH2-CHCH
FA CH3
Bonded Lys
FA CH2-CH2-CO
Bonded Ala
FA CH2
27
II. HRMAS-NMR of Lung Tumor Tissues
23
What can NMR tell us about human lung composition?
500 MHz 1H HRMAS-NMR
1D Standard T2 Edited (CPMG)
4.5 to 2.9 ppm
Glycerophosphocholine (GPC)
Phosphocholine (PC)
Choline (Cho)
Phosphoethanolamine (PE)
Gly
Myo-inositol (m-Ino)
m-Ino
GPC
Ethanolamine
Several aa
Lactate
Creatine
Lys, Tyr, Creatine
MeOH
GPC
PC
Taurine
28
II. HRMAS-NMR of Lung Tumor Tissues
24
What can NMR tell us about human lung composition?
500 MHz 1H HRMAS-NMR
1D Standard T2 Edited (CPMG)
Tyr
Phe
Ade, Ado, Hypoxanthine
Uracyl
Uridine
Uridine
Uracyl
29
II. HRMAS-NMR of Lung Tumors
How different are 2 fragments of the same tumor
sample?
30
I.2. Introdução RMN e a Investigação Oncológica
14
NMR Metabonomics of Hepatocellular Carcinoma
(HCC) J Proteome Res 2007
Bioquímica do HCC alterações no metabolismo
energético e no ciclo de Krebs
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