dha and cis-trans isomerization: applicability from chemistry to … · 2014-03-26 · dha and...

DHA and cis-trans isomerization:

Applicability from chemistry to

nutraceuticals

Giorgia Giacometti Doctor in Pharmaceutical Chemistry and Technology

Coffee Talk

March 2014, 18th

Summary of the Presentation

• Overview of the project

• 1st goal: Synthesis and Analysis

• 2nd goal: Diagnostics

• 3rd goal: Nutraceutical

• 4th goal: Biology

• Conclusions and perspectives

►Overview of the project

• 1st goal: Synthesis and Analysis






DHA: Docosahexaenoic acid

• 22 carbons chain and 6 double bonds in cis configuration

• Food sources: Fish and microalgae

• Nutraceutical sources: Fish and microalgae oils

• Biologic role linked to the all-cis configuration:

– Organs development and functionality: brain and retina

– Beneficial effects on the cardio-vascular system

– Precursor of metabolites with anti-inflammatory activity: neuroprotectins

DHA: Docosahexaenoic acid

• 22 carbons chain and 6 double bonds in cis configuration

• Food sources: Fish and microalgae

• Nutraceutical sources: Fish and microalgae oils

• Biologic role linked to the all-cis configuration:

– Organs development and functionality: brain and retina

– Beneficial effects on the cardio-vascular system

– Precursor of metabolites with anti-inflammatory activity: neuroprotectins

R 1 R 2 R 1

R 2

c i s t r a n s

Isomerization

Acc. Chem. Res., 2005, 38, 441–448 Chem. Rev., 2014, 114, 255–284

Cis-trans isomerization of DHA

Gruppo BioFreeRadicals

Dr. C. Ferreri; Dr. C. Chatgilialoglu

ISOF – CNR, Bologna

BIOLOGY:

Effect of the

membrane

organization

SYNTHESIS and

ANALYSIS:

Molecular

Library

Goals

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products

Cis-trans

isomerization

of DHA

R S + R 1

R 2

R S

R 1

H H R

2 R 1

R S + R 2

Formation of trans fatty acids

• Endogenous Processes − Radical reaction catalyzed by

sulfur-centered radicals (RS•)

All-cis isomer

R 1

R 2

R 1

R 2

R 1

R 2

R 1

R 2

R S R S

R S R S All-trans isomer

Mono-trans isomers

Step-by-step mechanism




• Industrial Processes – Partial hydrogenation

– Deodorization




mono-trans

DHA

• Industrial Processes – Partial hydrogenation

– Deodorization




DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products

Transesterification

Transesterification

Blood samples and nutraceutical products

Analytical Protocol for

Gas-Chromatography

Estraction

Estraction

Fatty acid

methyl esters

(FAME) RECOGNITION

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products

Synthetic Protocol:

mono-trans isomers

of Me-DHA

Transesterification

Transesterification


Gas-Chromatography

Estraction

Estraction

Fatty acid

methyl esters

(FAME)

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products


SYNTHESIS and

ANALYSIS:

Molecular

Library

Synthetic Protocols

in literature:

•mono-trans Me-LA

•mono-trans Me-ARA

• mono-trans Me-EPA

Synthetic Protocol:

mono-trans isomers

of Me-DHA

Transesterification

Transesterification


Gas-Chromatography

Estraction

Estraction

Fatty acid

methyl esters

(FAME)

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products


SYNTHESIS and

ANALYSIS:

Molecular

Library

Synthetic Protocols

in literature:

•mono-trans Me-LA

•mono-trans Me-ARA

• mono-trans Me-EPA

ANALYSIS and

RECOGNITION

by Gas-

Chromatography


Overview of the project

►1st goal: Synthesis and Analysis





SYNTHESIS and

ANALYSIS:

Molecular

Library

Goals (1)

Cis-trans

isomerization

of DHA

Mono-trans isomers of Me-DHA:

Synthesis and characterization

CHARACTERIZATION

• GC

• GC/MS

• 1H-NMR

• 13C-NMR

• COSY

• HSQC

1) 0,5 eq HOCH2CH2SH, iPrOH, hn

5 min, argon

2) Purification by Ag-TLC

Yield 30%

All-cis Me-DHA

Mix of mono-trans isomers of Me-DHA

Synthetic Protocol:

mono-trans isomers

of Me-DHA

Mono-trans isomers of Me-DHA

All-cis Me-DHA

GCTREND

1) 0,5 eq HOCH2CH2SH, iPrOH, hn

5 min, argon

2) Purification by Ag-TLC

Yield 30%

All-cis Me-DHA

Mix of mono-trans isomers of Me-DHA

Mono-trans isomers of Me-DHA:

Synthesis and characterization



1st goal: Synthesis and Analysis

►2nd goal: Diagnostics




SYNTHESIS and

ANALYSIS:

Molecular

Library

Goals (2)

DIAGNOSTICS:

Identification in

blood

samples

Cis-trans

isomerization

of DHA

8 blood samples

Lipid fraction

(PL, TG, CE e lipoproteins)

Fatty Acids Methyl Esters

(FAME)

Membrane phospholipids

of mature erythrocytes


(FAME)

GC Analysis and comparison with the library of mono-trans isomers of Me-DHA

0,5 M KOH in MeOH (30 min) 0,5 M KOH in MeOH (10 min)

Blood Plasma Blood Cells

H2O/Brine and CHCl3/MeOH 2:1

1) High-Throughput Procedure

(Patent of Lipinutragen srl)

2) H2O/Brine and CHCl3/MeOH 2:1

Centrifugation

Analytical Protocol: Blood Samples

Erythrocytes and plasma

Erythrocytes Mean (%) ± s.d.(n=8)

∑ 18:1 mono-trans 0,12 ± 0,03

∑ 18:2 mono-trans 0,16 ± 0,07

∑ ARA mono-trans 0,55 ± 0,15

∑ DHA mono-trans nd

Plasma Mean (%) ± s.d.(n=8)

16:1, 6-trans 0,05 ± 0,02

16:1, 9-trans 0,02 ± 0,01

∑ 18:1 mono-trans 0,09 ± 0,06

∑ 18:2 mono-trans 0,03 ± 0,01

∑ ARA mono-trans 0,04 ± 0,02

∑ EPA mono-trans 0,01 ± 0,01

∑ DHA mono-trans 0,02 ± 0,01




2nd goal: Diagnostics

►3rd goal: Nutraceutical



SYNTHESIS and

ANALYSIS:

Molecular

Library

Goals (3)

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products

Cis-trans

isomerization

of DHA

Lipid Fraction


(FAME)

H2O/Brine and CHCl3/MeOH 2:1

0,5 M KOH in MeOH (30 min)

GC Analysis and comparison with

the library of mono-trans isomers

of Me-DHA

Analytical Protocol: nutraceutical products

• 5 Supplements containing fish oil

• 1 Supplement containing algal oil

• 1 Raw material for functional foods

• 1 Powdered milk for infants

Principal families of fatty acids (%)

0 10 20 30 40 50 60 70 80 90 100

Saturi

Monoinsaturi

Polinsaturi omega 6

Polinsaturi omega 3

Acidi grassi trans

Supplement 1 (Fish Oil)

Powdered Milk (Vegetal Oil)

Raw Material (Fish Oil)

Supplement 6 (Algal Oil)





Saturated

Monounsaturated

Polyunsaturated omega 6

Polyunsaturated omega 3

Trans Fatty Acids

0 10 20 30 40 50 60 70 80 90 100

Omega 3 content (%)

56,14% of which DHA 22,6%

76,08% of which DHA 31,16%


48,62% of which DHA 48,28%

58,97% of which DHA 25,55%

43,98% of which DHA 16,13%


83,37% of which DHA 28,91%









0 10 20 30 40 50 60 70 80 90 100

Trans fatty acids content (%)









3,16% of which mono-trans DHA 1,38%


1,00% (mono-trans DHA NOT DETECTED)

NOT DETECTED





0 10 20 30 40 50 60 70 80 90 100













NOT DETECTED









3rd goal: Nutraceutical

►4th goal: Biology


BIOLOGY:

Effect of the

membrane

organization

SYNTHESIS and

ANALYSIS:

Molecular

Library

Goals (4)

DIAGNOSTICS:

Identification in

blood

samples

NUTRACEUTICAL:

Quality of

commercial

products

Cis-trans

isomerization

of DHA

Biomimetic studies on liposomes

• POPC (palmitic and oleic) and SDPC (stearic and DHA)

• Liposomes with 5% and 20% of DHA

• Extrusion technique to form unilamellar liposomes

• Irradiation by gamma-radiolysis in anaerobic conditions in the presence of 2-mercaptoethanol (HOCH2CH2SH)

H2O eaq

–(45%), HO•(45%), H•(10%) INIZIALIZATION

RSH + H•/HO• RS•

RS•

1. Influence of the phospholipid organization in membrane

2. Supramolecular organization: regioselective attack by thiyl radicals (RS•)

3. Reactivity of unsaturated fatty acids: oleic vs. DHA

4. Influence of the DHA concentration


Isomerization of DHA included in the

phospholipidic double layer of liposomes

Isomerization of DHA

in alcoholic solution



Time (min)

RS•

GC TREND




Liposomes in water

FAME in alcohol

RS•

GC TREND

Liposomes in water

FAME in alcohol




Trans-oleic

mono-trans DHA

Trans-oleic

mono-trans DHA

Liposomes with 5% of DHA

(Ratio oleic/DHA : 9:1)

Liposomes with 20% of DHA

(Ratio oleic/DHA : 3:2)

Time (min) Time (min)





Formation mono-trans of DHA in

liposomes with 5% of DHA

Formation mono-trans of DHA in

liposomes with 20% of DHA




4. Influence of the DHA concentration


Time (min)

Are

a m

on

o-t

ran

s is

om

ers

of D

HA

(%

)





3rd goal: Nutraceutical

4th goal: Biology

►Conclusions and perspectives

Conclusions

1. Expansion of a molecular library used as reference with

the synthesis of the mono-trans isomers of Me-DHA

2. Identification of mono-trans isomers of DHA only in the

plasma fraction of blood samples

3. Identification of mono-trans isomers of DHA in

nutraceutical products containing fish oil

4. Preliminary observations of the regioselective cis-trans

isomerization of DHA in biomimetic models of cellular

membranes

Perspectives

• Enantioselective synthesis of the

mono-trans isomers of DHA by

alternative strategies (Prof. Roberti,

University of Bologna)

• Studies on blood samples with

different pathologies (e.g. neurological)

• Computational simulation studies:

molecular dynamics and influence of

the cis-trans isomerism (Prof.

Recanatini, University of Bologna)

Sistem with POPC

(95%) and SDPC (5%)

Acknowledgement

• Dr. Carla Ferreri

• Dr. Chrys Chatgilialoglu

• Dr. Michele Melchiorre

• Dr. Anna Sansone

• Lipinutragen srl

• Prof. Maurizio Recanatini

• Prof. Marinella Roberti

ALMA MATER STUDIORUM

University of Bologna -

Department of Pharmacy

and Biotechnology

Thank you for your attention

dha and cis-trans isomerization: applicability from chemistry to … · 2014-03-26 · dha and...

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