Palmitoleic Acid and Algae Extract Collaboration S. Vanessa Aguilar Dr. Christine Schmidt Dr. Rhykka Connelly 11/16/2011

Yesterday we won intramural championship

GOALS Algae Extract (fatty acid) Cancer 1 Diabetes 2 Others applications 1 Kato, et al, Nutrition and cancer, 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 μM μM 1 1.Ruiz-Vela et al., Stem Cell Rev and Rep, Kato et al, Nutrition and Cancer, 2007

Control max Ethanol vehicle μM Control max DMSO vehicle 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 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 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

Controls Control Control DMSO (highest 5%) Control Ethanol (highest 5%) DMSO is killing cells.

Cell Viability 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 mg – Myristic acid – 248 μg (8.6%) – Palmitic acid – 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 mg – Carotenoids/xanthophills – Vitamins – Anything lipid soluble

Methods Control 1 Control max Palmitoleic Acid μM Control max Algae Extract 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

Algae Extract

Cell Viability

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 Tox Control 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 extract100 μM Algae extract 250 μM Algae extract

Palmitoleic acid day 3 Ethanol control 25 μM Palmitoleic acid 100 μM Palmitoleic acid 250 μM Palmitoleic acid

25 uM Algae extract 100 uM Algae extract 250 uM Algae extract Algae Extract day 3

Conclusions Qualitatively, there is an increase in lipids inside cells. Check pH of the testing media.