chemical imaging on liver steatosis

Chemical Imaging on Liver Steatosis Using Synchrotron

Infrared and ToF-SIMS Microspectroscopies

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Stéphane Nguyen

Plan

• Introduction

• Materials and Methods

• Results

• Discussion

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Introduction

• Steatosis: alcoholism, drug intake, small-bowel by-pass surgery or metabolic syndrome

• Non alcoholic fatty liver disease (obesity, insulin resistance, diabetes, drugs and the metabolic syndrome): most common cause of chronic liver disease in Western countries

• precursor for steatohepatitis: a condition that may progress to cirrhosis and in some cases to the development of primary liver cancer

• In countries with obesity: 25% of the general population and very common in overweight persons over the age of 30*

3 13/11/2012 *http://www.virtualmedicalcentre.com/diseases/fatty-liver-steatosis-steatohepatitis/648

Introduction

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DAG accretion

• Imbalance in the uptake, synthesis, export and oxidation of fatty acids

Formation of steatotic vesicles in the hepatocytes

Problems

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Primary metabolic abnormalities leading to lipid accretion

Local lipid composition

Materials and Methods: Patients and liver samples

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doi:10.1371/journal.pone.0007408.t001

• Tissues fixed in formalin

• One specimen of non-tumorous liver snap frozen in liquid nitrogen and stored at -80°C

Materials and Methods: Tissue section

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doi:10.1371/journal.pone.0007408.g001

PT: portal tract, BD: biliary duct, PV: portal vein, HA: hepatic artery, CLV: centrilobular vein, SV: steatotic vacuole.

• Normal hepatic lobule • fatty liver area : exhibiting macrovacuolar and microvesicular steatosis

X100

X400

Materials and Methods: Synchrotron FTIR microspectroscopy

• chemical composition in small sample area

• analysis at the diffraction while preserving a high spectral quality

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http://www.chemicool.com/definition/fourier_transform_infrared_spectrometer_ftir.html

Materials and Methods: ToF-SIMS Imaging

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• Ions are identified according to their mass-to-charge (m/z) ratio • Accessible mass range about 1500 Da • Ensured a spatial resolution of 1–2 mm and a mass resolution M/DM=104

http://www.geomuseum.uni-goettingen.de:8001/people/vthiel/tof_sims/index.shtml

Results: Chemical imaging on steatosis using

FTIR microspectroscopy

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Optical image of steatotic hepatocytes

Steatotic hepatocytes

Chemical imaging of some bands

Non-steatotic hepatocytes

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Second derivatives of IR spectra • slight downward shift of the CH2

symmetric and anti-symmetric stretching modes in steatosis vesicles

• lipids in a different environment, probably with higher structural order

Results: Chemical imaging on steatosis using

FTIR microspectroscopy

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• normal liver • non-steatotic area of fatty liver

Biopsies stained with HES (hematoxylin, eosin and safran) (x400)

FTIR spectroscopy on periportal hepatocytes

• higher intensity of CH3 and CH2 bands (2800–3000 cm-1) and esters (1710–1760 cm-1)

• Changes in the frequency domains 950–1200 cm-1 corresponding in part to sugar contribution

• small lipid droplets exhibiting sizes less than 10 mm containing DAGs

C=O

CH2-CH3

Results: Spectroscopic analysis of non-steatotic

hepatocytes in fatty liver

Results: Chemical imaging on steatosis using ToF-SIMS

mass spectrometry

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Positive ion mode video

• DAG C36 inside

• Overlay: DAG C36 and cholesterol co-localized in steatotic vesicles

MAG cholesterol

• DAG C30 outside of steatotic vesicles

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• DAG C30, C32, C34 and C36 bearing saturated alkyl chains: outside of steatotic vesicles

• DAG containing at least one unsaturated acyl chain: concentrated into steatotic droplets

Results: Chemical imaging on steatosis using ToF-SIMS

mass spectrometry

Discussion

• synchrotron source to study the complete frequency range from 900 to 4000 cm-1 (lipids)

• ToF-SIMS for investigating the local composition and distribution of the molecular species of lipids

• investigation at cellular and subcellular levels

• Concentration of unsaturated lipids inside steatotic vesicles may constitute a potential highly reactive site for peroxidation

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Perspectives

• infrared spectroscopy might be used as a diagnosis mean especially in the setting of liver transplantation

• spatial resolution and sensitivity of synchrotron FTIR microspectroscopy and mass spectrometry may open new avenue for characterizing early events in pathologies or for identifying markers for diagnosis and prognosis

• FTIR microspectroscopy using conventional infrared source might be set up in hospitals for clinical use

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chemical imaging on liver steatosis

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