Palmitoleic Acid and Algae Extract Collaboration

S. Vanessa AguilarDr. Christine SchmidtDr. Rhykka Connelly

11/16/2011

Yesterday we won intramural championship

GOALS

Algae Extract

(fatty acid)Cancer1

Diabetes2

Others applications

1 Kato, et al, Nutrition and cancer, 2007.2 Yang, et al, Lipids in health and disease, 2011.

Background

Using fatty acid-rich albumin in human embryonic stem cells (albuMAX) triggers adipogenic features.

Ruiz-Vela et al., Stem Cell Rev and Rep 2011.

Aims

Optimization of biological activity of palmitoleic acid and algae extract using human dermal fibroblasts and an adequate vehicle

Compare differences between palmitoleic acid and algae extract

Develop a protocol to look at the internalization of lipids within cells

Aim 1

Optimization of biological activity of palmitoleic acid and algae extract using human dermal fibroblasts and an adequate vehicle

Develop a protocol to look at internalization of lipids within cells

Compare differences between palmitoleic acid and algae extract

Background

• Cell viability assay for fatty acid treatment– Vehicle• Dimethylsulfoxide (DMSO)1

• Ethanol 2

– Concentration• 10 μM1

• 125 μM1

1. Ruiz-Vela et al., Stem Cell Rev and Rep, 20112. Kato et al, Nutrition and Cancer, 2007

Control Control

max

Ethanol vehicle

2.5 25 250

μMControl

max

DMSO vehicle

2.5 25 250

μM

Methods

• Seed cells 5,000 cells/well• Test palmitoleic acid concentration in

ethanol and dimethylsulfoxide (DMSO).

• Check for proliferation using CellTiter 96 kit (Promega) 1, 2 and 3 days with tested media

Palmitoleic Acid in DMSO

Control DMSO control (high)

2.5 uM in DMSO 25 uM in DMSO 250 uM in DMSO

0

0.5

1

1.5

2

2.5

Day 1Day 2Day 3

Abs

orba

nce

(490

nm

)

• DMSO itself is killing the cells 5% DMSO • 250 μM can’t be tested using DMSO• 2.5 μM and 25 μM of palmitoleic acid is safe

2.5 PA μM in DMSO

3 Days

25 PA μM in DMSO

250 PA μM in DMSO

Palmitoleic Acid in DMSO

Control DMSO (highest 5%)

Palmitoleic Acid in Ethanol

Control Ethanol Con-trol (high)

2.5 uM in Ethanol

25 uM in Ethanol

250 uM in Ethanol

0

0.5

1

1.5

2

2.5

Day 1Day 2Day 3

Abs

orba

nce

(490

nm

)

• 2.5 μM and 25 μM of palmitoleic acid is safe• 250 μM of palmitoleic acid cells are not proliferating and dying

3 Days

2.5 PA μM in Ethanol

25 PA μM in Ethanol

250 PA μM in Ethanol

Control Ethanol (highest 5%)

Palmitoleic acid in Ethanol

Cell Morphology

250 PA μM in Ethanol 3 days 20x

250 PA μM in Ethanol 3 days 10x

Cell Viability

Control Ethanol Control (high)

2.5 uM in Ethol

25 uM in Ethol

250 uM in Ethol

DMSO control (high)

2.5 uM in DMSO

25 uM in DMSO

250 uM in DMSO

0

0.5

1

1.5

2

2.5

Day 1Day 2Day 3A

bsor

banc

e (4

90 n

m)

2.5 uM and 25 uM of PA in both Ethanol and DMSO are safe to use with fibroblast in vitro

Conclusions

• DMSO is not a good vehicle for palmitoleic acid.

• Ethanol is a good vehicle for lipids.• There is a cell response with 250 μM

palmitoleic acid.

Aim 2

Find the range of palmitoleic acid and algae extract concentrations using human dermal fibroblast and the adequate vehicle

Compare differences between palmitoleic acid and algae extract

Develop a protocol to look at internalization of lipids within cells

Test algae extract• Algae extract was dried and weighed (58.7 mg)• Dr. Connelly ran a gas solid chromatography to identify the lipid

soluble components.– Results

• Fatty acids 2.864 mg – Myristic acid – 248 μg (8.6%)– Palmitic acid – 415.5 μg (14.5%)– Oleic acid – 85 μg (3%)– Palmitoleic acid – 1344 μg (47%)– Linoleic acid -200 μg (7%)– Linolenic acid- 96 μg (3.3%– Eicosapentaenoic acid -476 μg (16.6%)

• Other 55.83 mg– Carotenoids/xanthophills– Vitamins– Anything lipid soluble

Methods

Control 1 Control

max

Palmitoleic Acid

25 100 250

μMControl

max

Algae Extract

25 100 250

μM

Control 2

• Seed cells 2,500 cells/well• Test palmitoleic acid concentration in

ethanol and algae extract.• Check for proliferation using CellTiter

96 kit (Promega) 1, 3 and 7 days with tested media.

Ethanol vehicle

Palmitoleic acid

Control Ethanol Control (high)

25 uM PA in Ethanol

100 uM PA in Ethanol

250 uM PA in Ethanol

0

0.5

1

1.5

2

2.5

3

3.5

Day 1Day 3Day 7

Abs

orba

nce

(490

nm

)

Algae Extract

Control 25 uM Algae Ex-tract in Ethanol

100 uM Algae Ex-tract in Ethanol

250 uM Algae Ex-tract in Ethanol

0

0.5

1

1.5

2

2.5

3

3.5

Day 1Day 2Day 3

Abs

orba

nce

(490

nm

)

Cell Viability

Control Ethanol Control (high)

25 uM PA in Ethanol

100 uM PA in Ethanol

250 uM PA in Ethanol

25 uM Algae Ex-tract in Ethanol

100 uM Algae Ex-tract in Ethanol

250 uM Algae Ex-tract in Ethanol

0

0.5

1

1.5

2

2.5

3

3.5

Day 1Day 3Day 7

Abs

orba

nce

(490

nm

)

Conclusions

• Day 3 did not show increase of proliferation. – Checking on the images I took after adding MTS

assay, my MTS assay was killing the cells.• Day 7 there are no differences between controls, 25

μM, and 100 μM of Palmitoleic acid and 25 μM of algae extract.

Aim 3

Find the range of palmitoleic acid and algae extract concentrations using human dermal fibroblast and the adequate vehicle

Compare differences between palmitoleic acid and algae extract

Develop a protocol to look at internalization of lipids within cells

Methods

• After Cell titer 96• Fixed with 4% PFA at room temperature for

30°C minutes• Rinse 3x5 in PBS• Stained with LipidTox red Neutral stain

(Invitrogen) 1:200 in PBS following Invitrogen protocol

Controls Day 1

Control Cell Titer and Lipid ToxControl LipidTox only

Palmitoleic Acid Day 1

Ethanol control 25 μM Palmitoleic acid

100 μM Palmitoleic acid 250 μM Palmitoleic acid

Algae Extract Day 1

25 μM Algae extract 100 μM Algae extract

250 μM Algae extract

Conclusions

• Qualitatively, there is an increase in lipids inside cells.

• Check pH of the testing media.