Effects of protolichesterinic acid isolated from

Cetraria islandica on lipid composition in cultured

cancer cells evaluated using HPLC-MS/MS

Finnur Freyr Eiríksson

Båstad, 9. nov 2011

Outline

• Introduction

• Lichens

• HPLC-MS/MS Validation and results

• D-optimal screening on UPLC-MS/MS

• Palmitic acid

• Conclusions

Metabolic changes in cancer

• Energy metabolism

• Lipid metabolism

• Warburg effect: glycolysis in favour of oxydative

phosphorylation

• Autophagy

Therapeutic opportunities

• Cancer metabolism is a target for current drug development

• Fatty acid synthase (FAS) is highly expressed in most human

carcinomas

– Level of expression varies and determines sensitivity to FAS inhibitors

• Products of cycloxygenase (COX) and lipoxygenases (LOX)

pathways linked to carcinogenesis

– Inhibitors suggested as chemopreventive

• Metformin

– Reduced cancer risk and cancer-related mortality

Lichen compounds

• Several lichen species have been used in traditional medicine.

– Treatment for tuberculosis and tumours

• Symbiosis between fungi and algae

• Metabolites derived from lichens shown to have anti-

proliferative and cytotoxic effects

• Usnic acid

– Proton shuttle (energy metabolism and autophagy)

• Protolichesterinic acid

– 5- and 12-Lipoxygenase inhibitor

– Possible FAS inhibitor

Usnic acid

Protolichesterinic acid

Cetraria islandica

Aims

• Development of a sample preparation technique and LC-

MS/MS method for cultured cells

– Palmitic acid

– LTB4

– 12-HETE

– 5-HETE

• Effects of protolichesterinic acid on lipid metabolism

– Lipoxygenases and FAS

– Extraction of protolichesterinic acid from Cetraria islandia• Purification with preparative high-pressure liquid chromatography

Methods

• Protein precipitation

– Poor sensitivity

• Liquid-Liquid

extraction

– Low recovery

• Solid phase extraction

– Good sensitivity0

5000

10000

15000

20000

25000

0 10 20 30 40 50 60 70 80 90 100

Pe

ak A

rea

% Organic (Methanol)

Optimization of SPE

LTB4

12-HETE

5-HETE

HPLC-MS/MS

Tandem Quadrupole Quattro Ultima

HPLC 1525 Micro HPLC pump at 0.25 ml/min

Analytical ColumnColumn Temperature

Waters Xbridge C18, 3.5µ, 2.1 x 50mm40°C

Mobile Phase A: 0.1% formic acid : Acetonitrile (95:5 % v/v)

B: Acetonitrile (100 % v/v)

Injection volume 20 µL

Source temperatureDesolvation temperature

120°C450°C

Polarity ES-

Compound name: 5-HETE

Coefficient of Determination: R^2 = 0.994470

Calibration curve: 9.07943e-011 * x̂ 2 + 1.08205e-005 * x + -0.000171062

Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area )

Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

pg/mL-0 5000 10000 15000 20000 25000 30000 35000 40000

Re

sp

on

se

-0.000

0.200

0.400

0.600

pg/mL

Re

sid

ua

l

-10.0

0.0

10.0

20.0

Calibration CurvesCompound name: LTB4

Coefficient of Determination: R^2 = 0.998375

Calibration curve: 7.3493e-011 * x̂ 2 + 3.45628e-005 * x + 0.000296622

Response type: Internal Std ( Ref 6 ), Area * ( IS Conc. / IS Area )

Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

pg/mL-0 5000 10000 15000 20000 25000 30000 35000 40000

Re

sp

on

se

-0.00

0.25

0.50

0.75

1.00

1.25

1.50

pg/mL

Re

sid

ua

l

-10.0

-5.0

0.0

5.0

10.0

15.0

Compund: LTB4 R^2 = 0.998375

PBS buffer; 120 – 30.000pg/mL

Compund: 5-HETE R^2 = 0.994470

PBS buffer; 120 – 30.000pg/mL

Detection of LOX products

LTB4

5-HETE

12-HETE

LOQ and Blank matrix

Validation ResultsValidation

Parameters

Results

LTB4 12-HETE 5-HETE

Range 120 - 30.000 pg/mL

LOQ %

Accuracy 9.3 -3.0 2.2

Precision 5.4 12.7 9.0

Intra-Assay %

Accuracy -1.2 - 3.6 -7.0 - 4.7 -2.4 - 6.9

Precision 1.9 - 5.0 3.8 - 11.7 3.7 - 10.5

Recovery 64.9% 38.5% 33.5%

Stability Stable for;

Auto sampler At least 12 hr.

Quantification of LOX products in

Capan 2 cancer cells

Control EtOH Control 2.5 µg/mL PA 5.0 µg/mL PA

5-HETE 274,7 261,0 226,3 250,5

0,0

50,0

100,0

150,0

200,0

250,0

300,0

350,0

Cal

cula

ted

Co

nce

ntr

atio

n (

pg/

mL)

5-HETE

Control EtOH Control 2.5 µg/mL PA 5.0 µg/mL PA

LTB4 20,0 29,4 48,5 27,1

12-HETE 29,7 34,7 17,8 32,2

0,0

10,0

20,0

30,0

40,0

50,0

60,0

Cal

cula

ted

Co

nce

ntr

atio

n (

pg/

mL)

LTB4 and 12-HETE

Design of Experiments (DoE)

Objective

• Improve Sensitivity

• Lower Analysis Time

Responses

• Peak Area

• Peak height

• Retention time

D-optimal design

• Two qualitative factors

– Type of column

– Type of organic phase

• Five quantitative factors

• Modeling was performed with PLS (partial least squares)

Software: Modde 8.0, Umetrics AB

Experimental Screening

FactorsUPLC-MS/MS

Ranges

Gradient slope 1.0 - 4.0 min

Organic start 5 - 40%

Flow 0.45 - 0.65 mL/min

Type of solvent Acetonitrile or Methanol

Type of columnUPLC BEH C18, 1.7 µm, 2.1x100mm

UPLC CSH C18, 1.7 µm, 2.1x100mm

Capillary voltage 1 - 3.5 kV

Collision energy 10 - 20 eV

PLS components

0.0

0.2

0.4

0.6

0.8

1.0

Com

p1

Com

p2

Com

p3

Com

p4

Com

p5

R2 &

Q2

R2Q2

Loading scatter plot

-0.5

-0.4

-0.3

-0.2

-0.1

-0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

wc[

2]

wc[1]

Flow

Col

Org

Gra

Cap

Colum(CSH C18)

OrgP(MeOH)

Flow*Gra

Flow*Cap

Flow*OrgP(MeOH)

Col*Org

Col*Cap

Col*Colum(CSH C18)

Org*OrgP(MeOH)

Gra*Cap

Gra*OrgP(MeOH)

Colum(CSH C18)*OrgP(MeOH)

Re1Re2

Ar1~

He1~

He2~

Re8

Ar2~

Ar8~He8~

Regression coefficients scaled and centered

(peak area)

5-HETE 12-HETE

Summary Plot

2.32

2.34

2.36

2.38

2.40

2.42

2.44

2.46

2.48

1 2 3 4

Are

a 1

2-H

ET

E

Gradient slope

OrgP (ACN)OrgP (MeOH)

OrgP (ACN)

OrgP (ACN)

OrgP (MeOH)

OrgP (MeOH)

2.59

2.60

2.61

2.62

2.63

2.64

2.65

2.66

2.67

2.68

2.69

2.70

2.71

1 2 3 4

Are

a 5

-HE

TE

Gradient slope

OrgP (ACN)OrgP (MeOH)

OrgP (ACN)

OrgP (ACN)

OrgP (MeOH)

OrgP (MeOH)

MODDE 8 - 06/11/2011 13:13:48

4-D countorplot (Area)5-HETE 12-HETE

Detection of Palmitic acid

Conclusions

• HPLC-MS/MS method was developed and validated

for quantification of LOX-pathway products at Pico-

gram levels

• Quantification of 5-HETE was successful in capan 2

pancreatic cancer cells

• The DoE was useful for evaluating factors affecting

responses of the method

• Palmitic acid not quantifiable because of

contamination problems

Bioprospecting from Icelandic sea

Acknowledgments

• Funding

– University of Iceland Doctoral Fund

– University of Iceland Research Fund

– ArcticMass

• Margrét Þorsteinsdóttir

• Baldur Bragi Sigurðsson

• Helga Ögmundsdóttir

• Kári Skúlason

• Sesselja Sigurborg Ómarsdóttir www.ArcticMass.is