The lipid content of human glioma cell

The lipid content of human glioma cell

ABSTRACT

The lipid content of human glioma cell line 1321n1 was analysed. A comparison of confluent and non-confluent tumour cells was undertaken for fatty acids, phospholipids and cholesterol concentration. After having undergone lipid extraction, fatty acids were measured using gas chromatography, phospholipids were separated out via thin layer chromatography and the free cholesterol concentration was measured using an enzymatic assay. The free cholesterol concentration had reduced in the confluent tumour cells compared to the non- confluent cells. Differences were found between the fatty acid methyl ester compositions in the confluent and non- confluent cells. An increase of the stearic and linoleic fatty acids was seen and a decrease in pentadecanoic acid was observed. Sphingomyelin was present in the confluent glioma cell line. Phosphatidylcholine and phosphotidylserine were present in both the confluent and non- confluent tumour cells. Hence, it can be said that there is a difference between the lipid compositions of confluent and non- confluent cells, which may aide in the identification of the lipid components that are potentially involved in the regulation of the proliferation of human glioma cell lines.

1 INTRODUCTION

1.1 Overview of glial cells

A glial cell is commonly referred to as a neuroglia. They are non- neuronal cells that provide support and nutrition, maintain homeostasis, form myelin and participate in signal transmission in the nervous system (Azevedo,2009). Myelin consists of 80% lipids and has many layers of a specialised cell membrane around the axons of particular nerves (Rosen, 2009). There are a great variety of glial cells in the central nervous system (CNS). There are four types, ependymal cells, astrocytes, oligodendrocytes and microglia which can be seen in the table below (Martini, 2006).

Table 1.1: The different types of glial cells present in the central nervous system.

Type of glial cell

Year of discovery

Structure

Function

Ependymal cell

Late 1990s

(Frisen, 2008).

On the walls of brain ventricles they play an essential role in the transport of CSF and in brain homeostasis (Spassky, 2005).

Astrocytes

1893

(Nash, 2010)

Maintains the blood brain barrier, creates a three dimensional framework for CNS, restores damaged neural tissue, a guiding neuron development and controlling the interstitial environment (Martini, 2006).

Oligodendrocytes

1921

(Mo, 2006).

Oligodendrocytes are involved together with Schwann cells in producing the myelin sheath, which coat the axons of nerve cells to speed up conduction and allow salutatory conduction to take place (Kandel, 2000).

Microglia

1919

(Friedman, 2009).

Microglia function as phagocytes that respond when needed to remove damaged tissue after surgery (Streit, 2002).

They also have the ability to migrate into the CNS to areas that undergo changes due to an injury.

Firstly, ependymal cells extend along the longitudinal axis of the spinal cord and the brain. The passage way consists of cerebrospinal fluid (CSF). A single layer of cells that is either cuboidal or columnar in shape is formed. These cells also possess microvilli and cilia (Snell, 2009).

In many areas of the brain, the passage way forms enlarged chambers called ventricles. The central canal and ventricles together are lined by ependymal cells, forming an epithelium known as ependymal (Martini, 2006). On the walls of brain ventricles, they play an essential role in the transport of CSF and in brain homeostasis (Spassky, 2005).There are three groups of ependymal cells; ependymocytes, tanycytes and choroidal epithelial cells (Snell, 2009).

Ependymocytes have contact with the cerebrospinal fluid (CSF) as they line the ventricles of the brain and also line the central canal of the spinal cord (Snell, 2009). The tanycytes allow ‘long basal processes to pass between the cells of the median eminence and the plate feet on the capillaries' (Snell, 2009). The choroidal epithelial cells have many folds in it which are held by ‘tight junctions'. These tight junctions stop the CSF from ‘leaking into the underlying tissues' (Snell, 2009).

Astrocytes are the largest and most common neuroglia in the CNS. This type of Glial cell has many functions such as maintaining the blood brain barrier, creating a three dimensional framework for the CNS, restoring damaged neural tissue, guiding neuron development and controlling the interstitial environment(Martini, 2006).

Astrocytes exist in 2 forms. These are fibrous and protoplasmic. The fibrous form of astrocytes, are usually found in great numbers in the white matter of the brain (Blows, 2002). They undergo a process called gliosis, when there is brain tissue damage. In this process they increase in; number, size and glial fibrillary acidic protein (GFAP) immunoreactivity (Cunha, 1993). The fibrous form have long cellular processes but they are fewer in number than the protoplasmic form (Blows, 2002).

The protoplasmic form of astrocytes is usually found in the grey matter of the brain. They have many short cellular processes and are extensively branched (Blows, 2002). Both the fibrous and protoplasmic forms have the ability to undergo mitosis at any time in an individual's lifetime, which many neurons cannot do (Blows, 2002).

The cell body of oligodendrocytes is much smaller and has fewer processes than astrocytes. Oligodendrocytes are involved together with Schwann cells in producing the myelin sheath, which coat the axons of nerve cells to speed up conduction and allow saltatory conduction to take place (Kandel, 2000).

The least numerous and smallest glial cell is the microglia and has the ability to migrate through neural tissue. As the nervous system is forming, microglia moves into the CNS (Martini, 2006). After injury, disease or infections, microglia that are phagocytes become mobilized (Kandel, 2000).

However, glial cells in the peripheral nervous system (PNS) consist ofperineuronal satellite cells which surround the cell body and Schwann cells that surround nerve fibres (Noback, 2005).

1.2 Lipid Rafts

Lipid rafts also known as detergent- resistant membrane domains are mainly composed of sphingolipids and cholesterol which reduces membrane fluidity (Chung, 2007). They are known as detergent- resistant membranes because when certain detergents where added to the cell membrane to solubilise it, they remained intact (Murphy, 2008). The side chains of the phospholipids on the lipid raft membranes are highly enhanced with saturated fatty acids. This allows the lipids to be closely packed within the raft (Calder & Yaqoob, 2007). Lipids play an important and diverse role in cells. The main functions that lipids carry out are the storage of chemical energy, provisional support of biological membrane and signalling. All have relevance to cells that undergo transformation, cancer progression and metastasis (Fernandis & Wenk, 2009). The synthesis of cholesterol in the endoplasmic reticulum is a process whereby the formation of lipid rafts begins and this is completed in the Golgi apparatus. This is followed by the membrane structures travelling towards the cell periphery. At the cell surface or via endosomes to the Golgi, lipid rafts can re-cycle through up regulation(Di Vizio, 2008).

Key

A

Intracellular space or cytosol

B

Extracellular space

1

Non-raft membrane

2

Lipid raft

3

Lipid raft associated transmembrane protein

4

Non-raft membrane protein

5

Glycosylation modifications

6

GPI-anchored protein

7

Cholesterol

8

Glycolipid

There are 2 types of lipid raft: Morphologically flat rafts which are occasionally referred to as G domains and Caveolae (Chung, 2007). Caveolae appear as ‘flask-shaped invaginations' of the plasma membrane and these lipid rafts have a protein coat of Caveolin around them (Fielding, 2006). They are present in adipocytes, myocytes, osteoblasts and endothelia (Deurs, 2003).

Functions of caveolae lipid rafts:

  1. Aid in the transport of molecules to and from the plasma membrane
  2. Aid in the transport of molecules between cellular compartments
  3. Compartmentalising in signalling pathways (Chung, 2007).

However, flat rafts are known to not contain Caveolin proteins and therefore membrane vesicles are not produced (Di Vizio, 2008).

In addition, rafts perform certain roles in processes such as membrane fusion, organisation of the cytoskeleton, lipid sorting and protein recycling (McIntosh, 2007).

Some proteins that are associated with lipid rafts are GPI-anchored protein and src family kinases. The functions of GPI (glycan phosphatidyl inositol) anchored proteins include enzymatic activity, protein- protein interactions, differentiation markers and membrane receptors (Young & Moss, 2000). Other membrane proteins do not become associated with rafts until they are oligmerised (Simons & Ehehalt, 2002).

Src family kinases activate further signalling pathways which includes a raft component called phosphatidyl inositol. This plays two important signalling roles when phosphorylated. Phospholipase C is broken down to give 2 second messenger molecules, which are inositol triphosphate and diacylglycerol (Murphy, 2008).

Reaction Scheme of sphingomyelin

When sphingomyelin is synthesised diacylglycerol is produced. This plays an important part in protein kinase C activation and is also involved in cell proliferation and signalling (Cerbon, 2009). Cholesterol-sphingomyelin rafts are dependent on the amount of membrane cholesterol and sphingomyelin. A reduction of either component contributed in effectively treating raft related diseases and infections (Yechezkel, 2004).

Phosphatidylcholine is hydrolysed by phospholipase D to generate phosphatidic acid (Foster, 2009). The mammalian targets of rapamycin mediated signals that promote cancer cell survival are regulated by phosphatidic acid (Foster, 2009).

The functions of cholesterol are to serve as a spacer between hydrocarbon chains of sphingolipids and to maintain the rafts assembly together (Simons & Ehehalt, 2002). Between the raft and the non-raft phase cholesterol separates. This results in cholesterol having a higher affinity to raft sphingolipids then to unsaturated phospholipids. If the raft cholesterol is removed, most proteins become non-functional (Simons & Ehehalt, 2002).

Cholesterol accumulates in solid tumours. Cholesterol feedback inhibition mechanism that regulates cholesterol synthesis is reduced in malignant forms of brain tumours (Gliemroth, 2003).

1.3 Human glioma cell line

There are two forms of cancer, benign and malignant. The production of tumours arises because cells no longer have the ability to create tissues of a normal form and function (Weinberg, 2007). A brain tumour is a condition where there is an abnormal growth of brain cells (McAllister,2002).Tumours that do not invade close tissues and only grow locally are classified as benign. However a malignant astrocyte is the most common primary brain tumour in adults and does invade nearby tissues (Weinberg, 2007). It is categorised into two types which are Glioblastoma multiforme and Anaplastic astrocytomas. Glioblastoma and anaplastic are classified as grade four and grade three astrocytomas (McAllister,2002).

Gliomas are a diverse group of aggressive glial tumours. They are the most frequent in the central nervous system, including sites in the brain and the spinal cord. This is followed by neuroblastomas, medulloblastomas, ependymomas and menigiomas (Herberman & Mercer, 1990). The 1321n1 human glioma cell lines were either confluent or non confluent. Non confluent is defined as rapidly dividing tumour cells where as confluent means that the cells are not dividing (REF).

1.3.1 Extraction of lipids

The aim of the experiment is to extract the lipids from the human glioma cell lines as complex acylated lipids in order for FAME (fatty acid methyl ester) analysis to be conducted by gas chromatography, thin layer chromatography of phospholipids and cholesterol assay.

The Folch method is used for lipid extraction which is one of the most common methods used around the world. It utilises chloroform and methanol in a 2:1 ratio (Rose & Klander, 1965).

Extraction of lipids will be conducted for the Human Glioma Cell Lines 1321n1 that consist of a passage of P13 and P14 that have undergone the cell cycle 13 or 14 times. It is an astrocytoma that was removed from a patient (Sigma- Aldrich, 2010).

1.3.2 Cholesterol

The total amount of cholesterol that resides in the brain is approximately 25% of the total amount of cholesterol in the body (Björkhem & Meaney, 2004). The role of cholesterol in the brain involves providing support of the plasma membrane and it is also involved in the proliferation of glioma cells (Maletínská, 2000). Cholesterol exists as cholesterol esters and also as free cholesterol. The free cholesterol concentration decreases in brain tumours, on the other hand, the concentration of cholesterol-esters increases (Köhler, 2009). The free cholesterol concentration can be determined by using the enzymes horseradish peroxidase and cholesterol oxidase. By adding cholesterol esterase, cholesterol- esters can be hydrolysed to produce cholesterol and then can be measured. The cholesterol oxidase oxidises the free cholesterol to produce cholest- 4- en-3- one as well as hydrogen peroxide, the latter of which oxidatively couples with 4- aminophenazone and phenol. This produces a quinoneimine dye that can be measured at a maximum wavelength of 500 nm (Allain, 1974).

1.3.3 Fatty acid methyl esters

The extraction of lipids from the human glioma cell lines as complex acylated lipids will be conducted in order for fatty acid methyl esters to be determined via gas chromatography. Fatty acids are monocarbonic acids that have chain length between 2 and 26 (Kohlmeier, 2003). Fatty acids have many uses in the brain such as they provide energy within all cells. An increase of fatty acid synthesis and use contributes to the growth of a brain tumour cell (Stimson, 2010).

Analysis of fatty acid methyl esters is carried out by Gas chromatography (GC) which is a technique that separates chemical substances. ‘This is achieved under the differences in separation between a flowing mobile phase and a stationary phase to separate components in a mixture' (Linde, 2008). Methyl esters are known to provide quick and quantitative samples for GC analysis.

Pentadecanoic acid is used as an internal standard and allows quantification. It has a molecular name of C15H30O2. It is not present in eukaryotic cells. Linoleic and stearic are the fatty acid methyl esters which will be used to determine the retention times of key fatty acids so then the fatty acid profiles of the lipid samples can be figured out. Stearic acid has the scientific name of octadecanoic acid and has a molecular name of C18H36O2. It is a saturated fatty acid (Myers, 2007). Linoleic acid has a molecular name of C18H36O2 (Youdim, 2000). Linoleic acid is a polyunsaturated fatty acid (Harrison, 2007).

For the FAME analysis a non polar GC column is used which is proven to be of great success in regards to separation. Gas chromatography is highly recognised due to its simplicity, high sensitivity and its excellence in separating components in a mixture. It is one of the most essential tools in chemistry (Linde, 2008).

GC consists of many different types of detectors but for this process a flame ionisation detector is used for mass flow and hydrogen and air are its support gases (Punrattanasin & Spada, 1997). To determine and document the concentration of separated constituents at the end of a column, a sensitive detector is required (Baugh, 1993).

The enhancement of volatilisation, separation, detection and thermal stability is due to the derivatisation of analytes. Organic acids, amides, poly-hydroxy compounds and amino acids are materials that need derivatisation. The 3 processes involved are esterification, acetylation and silylation (Lecture slide, Carole Rolph, 2009).

Another type of gas chromatography is the gas solid chromatography (Baugh, 1993). Gas solid chromatography is used when the mobile phase is a gas and the stationery phase is a solid (Berezkin, 1991).

The results from the samples eluted from the column will be compared to the retention time of the standard solutions of known fatty acids. The area under the peak will be evaluated under the integrator output and the fatty acid composition will be calculated for all the major peaks produced. Thus the fatty acid methyl esters that are present more and have an effect on proliferation in the confluent and non confluent cells will be identified and the effectiveness of the technique will be determined.

1.3.4 Phospholipids

Phospholipids in the lipid sample were separated by a process of thin layer chromatography. Thin layer chromatography (TLC) can be used to separate many organic compounds and is carried out in a tubular distribution system. Its success is due to its widespread applicability, its high resolving power, high capacity and the simplicity and controllability of the method. It consists of a mobile phase and stationery phase which consists of a silica gel (Anon, 2008). This is a layer that is spread over a flat plate, as the layer is thin a simple capillary action enables the movement of the mobile phase rapidly allowing a minimum amount of resistance to flow. The stationary phase is immobilised on a glass or plastic plate and an organic solvent (Anon, 2008). The sample is deposited as a spot on the stationary phase in either a liquid or dissolved state. The unknown samples are run at the same time as the constituents therefore it is easily identified as to what the unknown sample consists and allows variation (Lecture slide, Carole Rolph, 2009). However the tank requires equilibrating before use as a number of components are volatile within the mobile phase and therefore stops evaporation from the surface to the plate in order to sustain the mobile phase composition. When oppositely charged molecules are attracted to polar and non-partially molecules, consequently separation occurs. However absorption or binding will only obtain if the solutes of the stationary phase are polar than them of the mixture, requiring a good strength of the adsorptive bonds which reflects ionic interaction that take place. “The rate at which solutes move along the chromatogram is determined on the solubility of the mobile phase and the strength of any adsorptive bonds formed with the stationary phase.” (Lecture slide, Carole Rolph, 2009). The solubility of the mobile phase and the strength of any adsorptive bonds formed with the stationery phase determine the rate at which solutes move along the chromatogram. As the mobile phase (solvent front) reaches the end of the glass plate or when the capillary action comes to halt, the analyte movement stops. Analyte movement also stops if or when the plate is removed from the mobile phase reservoir (the tank) (Lecture slides, Carole Rolph, 2009). Spray reagents specifically used for TLC are introduced for example ninhydrin which consists of anilonapthosulphuric acid (ANSA) (solid) made up in anhydrous methanol (liquid) making it possible to view the results without difficulty as the compounds develop fluorescent and are easily examined under ultraviolet light (UV). The retardation factor (Rf) can be calculated which indicates the movement of the mobile phase (solvent front) with the movement of the analyte. The results can be identified by comparing 1321n1 lipid samples with the standards that were spotted and therefore indication of which phospholipids are present in the 1321n1 tumour cell lines.

The known standards that are used for the experiment are phosphotidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In cell membranes, phosphotidylcholine (PC) is the main element (Anon a). Maintaining the permeability barrier and providing a structural support is its key primary role involving in membrane mediated cell signalling (Anon a). Lecithin is a basic lipid that is found in animal and plant tissues and is known to be an essential factor in phosphotidylcholine. Lecithin is recognised to contain a range of diverse lipids. Phosphatidylinositol is a component of cell membranes that is known to act as a substrate for numerous enzymes that are involved in cell signalling. Phosphorylation occurs by a variety of kinases to form phosphatidylinositolphosphates (PIP) (Anon b). Phosphatidylethanolamine (PE) is a phospholipid which is also recognized as cephalin (Anon c). It is mainly found in biological membranes, above all in the nervous tissue. Approximately half of the PE is present in both the vinyl ether and half of the phospholipids content is made up of PE in the brain tissue. But the significance the statement holds is not yet known (Anon c). Generally animal tissues contain approximately 1-2% of phosphatidylglycerol (PG) as a basic constituent of cell membrane (Anon d). However the significant levels of PG add up to 11% in the lungs of the whole lipid content (Anon d). Sphingomyelin is a lipid that consists of mainly fat content in the cell membranes. A polar group which is made up of phosphoethanolamine or phosphocholine, a fatty acid which is usually saturated and a sphingosine is the basic structure of a sphingomyelin (Martin 2003).

1.4 WORKING HYPOTHESIS

This project tests the hypothesis that the lipid components that are potentially involved in the regulation of the proliferation of the human glioma cell line 1321n1 can be characterized in confluent and non- confluent cells.

1.5 AIMS OF THE INVESTIGATION

To quantify and characterise the lipid components potentially involved in the regulation of the proliferation of the human glioma cell line 1321n1.

For each experiment both confluent and non confluent cultures were used to analyse the differences between them as non confluent cells are rapidly dividing which is associated with cell proliferation, where as confluent cells are not actively dividing and have reached their maximum ability.

  1. To determine the free cholesterol concentration via an enzymatic cholesterol assay.
  2. To extract the fatty acids as complex acylated lipids but to analyse as fatty acid methyl esters via the process of gas chromatography.
  3. To determine the types of phospholipids via thin layer chromatography.

2 MATERIALS AND METHODS

Cell culture

1321n1 human glioma cell line was used and stored in the freezer at a temperature of -2oC.

  • Were it came from
  • Grown and harvested

Extraction of Lipids

Lipids were extracted from 1321n1 human glioma cell lines which were confluent and non-confluent using the Folch method. Into a 100 ml conical flask 5 g of NaCl was added and distilled water. This was then overted a couple of times. Into a methylating tube containing the 1321n1 confluent cells 4 ml of chloroform and 2 ml of NaCl was added (2:1, v/v) and this was allowed to separate into two phases. The chloroform phase was removed carefully into another methylating tube and in a fume cupboard this was blown down to dryness under nitrogen. Around the sides of the tube pure chloroform was rinsed and when dried the mobile phase was put into the methylating tube. The process was repeated for the 1321n1 non-confluent cells. The lipids were visualised around the sides of the methylating tube once pure chloroform was added.

Cholesterol Assay

A 3 X solution of the reagent mixture was prepared using 32.3 mg Sodium cholate, 2.9 mg 4- Aminophenazone, 47 mg Phenol, and 4 µl Triton X-100. This was then diluted 1in 3 by 0.1 M Tris HCl buffer which was prepared to pH 7.4. 100 µl of horseradish peroxidase was then added to the mixture along with 6 µl of cholesterol oxidase.

The cholesterol stock solution was then prepared to a concentration of 25.6 mmol/l using, 100 mg of Cholesterol (sigma 99 % grade),10 ml of ethanol diluents (absolute ethanol containing 20 % Triton X-100).

The dilutions of cholesterol stock solutions along with the confluent and non-confluent glioblastoma samples used can be seen in the appendix, Table 1.

The absorbance was measured at wavelength 492 nm on the GeniousPro microplate reader.

Preparation and extraction of FAMES

High grade (Analar) MeOH was placed over anhydrous sodium sulphate so then the MeOH would become anhydrous. On the day of the preparation, 50 mls of anhydrous MeOH was filtered into a conical flask. . In the fume cupboard 1.25 mls of concentrated sulphuric acid was added and this was swirled to mix the components. This is known as the methylating solution. The lipid samples were re-dissolved in 0.2 ml chloroform and placed into a methylating tube and then blown down to dryness under nitrogen. After this, 2 mls of methylating solution was added. Then the tubes were placed in the heating block at 70oC for 2 hours and after this cooled down to room temperature. The methylating tubes were then stored in the freezer until required.

In regards to extraction, 50ml 5% NaCl solution was made up and 5ml was added into each of the methylating tubes. 12.5 mg/ml pentadecanoyl methyl ester in petroleum ether was made up and 10 µl was added to each sample using a GC syringe. This was the internal standard and allowed quantification. Then 3 ml of petroleum ether (Analar grade) was added and vortexed so then the contents would be mixed. The upper phase was removed into a conical glass centrifuge tube. The same was repeated to combine the upper phases in the same conical centrifuge tube and this was blown down under nitrogen until there was approximately 1 ml of extract left in the tube. The remaining was then blown down to complete dryness and the sample was re-dissolved in no more than 30µl of petroleum ether. Into the GC which was called, 2µl was injected.

Name of GC!

The gas chromatography was run for an official time of 20 minutes for the standards and for the samples, 40 minutes. The analytical conditions were that the oven was maintained at a temperature of 100oC and the GC was temperature programmed to 50oC and was increasing by 12oC every 1 minute up to the final temperature of 240oC.The injector temperature was 50oC and detector was 250oC respectively.

Info about GC column

The fatty acid methyl esters were identified by comparison of the retention times with the standards.

Thin layer chromatography

A G60 silica gel plate was put in the oven between 100oC- 150oC for 2 hours. The following solvent was made up using chloroform (85mls), methanol (15mls), acetic acid (10mls) and distilled water (3.5mls). A TLC tank was first lined up with Whatman No 1 filter paper. The solvent was poured in and the lid replaced. It was left in the fume cupboard for 1 hour. The longer the tank equilibrates, the more accurate the results. The lipid sample was re-dissolved in 0.2mls of chloroform and was blown down to dryness under nitrogen. Once the lipid sample was dry, 0.05mls of chloroform was added. The silica gel plate was taken out of the oven and left to cool. A 2cm line was drawn up from the bottom of the plate which marks the origin. The standards, sphingomyelin were spot along with the lipid sample (P13 confluent and non-confluent and P14 confluent and non-confluent) at 1.25cm apart on the line of the origin which allows an adequate amount of liberty for the lipids to travel. Once the solvent had evaporated, the TLC plate was placed in the tank with the lid on correctly thus no evaporation would occur. Once the solvent had travelled to 75% of the plate, it was important the plate was continuously checked as this was the critical stage. If the solvent stopped moving then the samples would bunch up. When the solvent had reached 2.5cm from the top of the plate it was removed and the solvent front was marked and left to dry in the fume cupboard. Once dry a spray of 0.025% of Anilinonaphthosulphonic acid (ANSA) and 100mls of anhydrous methanol was prepared. The plate was viewed under the UV light and all the bands appeared were marked with a pencil so the bands can be viewed at a later date.

3 RESULTS

3.1 Cholesterol Assay

From Figure 3.1.1, it can be seen that as the free cholesterol concentration (mmol/l) increased, the absorbance values measured at wavelength 492 nm also increased. This was true for the standards after 1, 2 and 3 hours of incubation at room temperature.

At 5 mmol/l concentration of cholesterol, the absorbance value measured after 1 hour of incubation (at room temperature) was 0.044. After 2 hours of incubation, the absorbance measured at 5 mmol/l concentration had increased to 0.067 and after 3 hours of incubation the absorbance had further increased by 0.0152 to 0.0825. This showed that after a few hours at the same concentration, the absorbance value measured had increased.

At 15 mmol/l of cholesterol concentration, the absorbance value measured after 1 hour was 0.125. After 2 hours the concentration had increased by 0.079 to 0.204 and had increased again after 3 hours by 0.039 to 0.243.

At 20 mmol/l of cholesterol concentration, the absorbance value measured after 1 hour was 0.154. It had increased by 0.12 to 0.274 at 2 hours of incubation. It had further increased by 0.049 to 0.323 at 3 hours.

It can be seen from the results that at a lower concentration (mmol/l), the absorbance value measured increased more after the assay was left for incubation for 3 hours.

From Table 3.1.3 it can be seen that the absorbance values for the confluent samples P13 and P14 were lower in comparison to the non- confluent samples P13 and P14 samples. There was a larger difference between the values of the P14 confluent and non- confluent samples.

3.1.3 Table to show the mean absorbance values measured at wavelength 492nm.

Absorbance- 1 hour

Absorbance- 2 hours

Absorbance- 3 hours

Confluent P13

0.1239

0.1272

0.1021

Non-Confluent P13

0.2922

0.3048

0.3106

Confluent P14

0.1298

0.0862

0.0985

Non-Confluent P14

0.2942

0.3003

0.3123

The cholesterol concentration decreased in the confluent lipid sample compared to that of the non- confluent sample. This is shown in Table 3.1.4.

3.1.4 Table to show the mean cholesterol concentration (mmol/l) of the confluent and non-confluent cells.

Concentration- 1 hour (mmol/l)

Concentration- 2 hours (mmol/l)

Concentration- 3 hours (mmol/l)

Confluent P13

15.2

9.5

6.2

Non-Confluent P13

-

22.6

19.6

Confluent P14

15.8

6.5

5.9

Non-Confluent P14

-

22.4

19.4

3.2 Preparation and extraction of FAMES for GC analysis

Figure 3.2.1 shows that at retention time of 2 minutes a solvent peak was produced and this indicated that the sample was running effectively. At a time of 13.38 minutes the peak was identified to be Pentadecanoic acid methyl ester, 16.70 minutes was Linoleic acid methyl ester and at 15.85 Stearic acid methyl ester.

From Figure 3.2.2 it can be seen that at retention time of 11.67, 14.45 and 16.49 minutes, the fatty acids palmitic (16:0), linoleic (18:2) and stearic (18:0) are present. The identification of the chromatographic peaks was performed by comparing with known standards. In the confluent sample there are many other fatty acids present. The glioma content of linoleic and stearic acid is greater in the confluent sample in comparison to the non confluent sample by 1.81% and 5.86%.

Figure 3.2.3 shows that the peaks produced were of similar retention times of the standard fatty acid methyl esters but here palmitic acid was identified at a retention time of 11.40 minutes. However there are fewer peaks of fatty acid methyl esters present in comparison to the confluent sample.Thus the fatty acid composition does differ in a non confluent glioma cell line.

Figure 3.2.4 shows two major peaks which are identified to be linoleic (18:2) and stearic acid (18:0) at retention times of 14.16 and 15.91 minutes. However at retention time of 11.56 minutes there is palmitate present.

3.3 Thin Layer Chromatography for the separation of phospholipids

Looking at the Rf values obtained it can be seen phosphatidylcholine, phosphatidlyserine and cholesterol are present at different points on the TLC plate. Therefore there is a difference in the Rf values for both confluent and non-confluent P14 lipid samples. As the table shows other phospholipids were also present in the non-confluent P14 lipid sample at a very high Rf value. It can also be seen that a high intensity of cholesterol was present in the p14 non-confluent tumour cell lines as to the confluent tumour sample. However there were other lipids present, which could have been cholesterol as it is known to be present in confluent and non-confluent cells. There are other lipids present at the stationary phase.

3.1.1 The mean absorbance measured at 492 nm after 1, 2 and 3 hours of incubation.

Cholesterol concentration (mmol/l)

Absorbance- 1 hour

Absorbance- 2 hours

Absorbance- 3 hours

25.6

0.1401

0.2623

0.3122

18.2

0.0378

0.0979

0.1430

15.6

0.1525

0.2376

0.2814

13.0

0.0797

0.1611

0.1927

10.4

0.0939

0.1445

0.1610

7.8

0.0774

0.1176

0.1359

5.2

0.0539

0.0686

0.0782

2.6

0.0462

0.0495

0.0401

0.0

0.0011

-0.0082

-0.0068

3.1.2 Table to show the mean absorbance of the quality control measured at wavelength at 492nm after 1, 2, and 3 hours.

Cholesterol concentration (mmol/l)

Absorbance- 1 hour

Absorbance- 2 hours

Absorbance- 3 hours

6.5

0.0677

0.1262

0.0869

3.2.1 The standard results for the fatty acid methyl esters

The standards consisted of Pentadecanoic, Linoleic and Stearic acid methyl esters. Using a GC syringe 2µl of the standard samples was injected into the gas chromatography column.

3.2.1 Table to show the results obtained from the standard samples.

Standard samples

Carbon chain length of fatty acid

Retention time for each FAME (minutes)

Pentadecanoic acid methyl ester

C15

13.38

Linoleic acid methyl ester

C18

16.70

Stearic acid methyl ester

C16

15.85

From the gas chromatograph 3 peaks were produced showing the three standards.

From Table 3.2.1 it can be seen that the saturated fatty acids were extracted first followed by the polyunsaturated fatty acids. This is due to that Pentadecanoic fatty acid methyl ester had the shortest retention time of 13.38 minutes in comparison to Linoleic and Stearic acid methyl esters. The samples were injected into the gas chromatograph and run for 40 minutes.

3.2.2 The fatty acid composition for the confluent sample.

The sum of the total area mV* (milli volt) minute is 113.119. Refer to appendix for calculations.

Retention time (minutes)

Carbon chain length of fatty acid

Area mV* min

Percentage of fatty acid composition (%)

11.67

16:0

20.825

18.41

14.45

18:0

30.285

26.77

14.69

18:1

15.243

13.475

16.49

18:2

36.434

32.21

17.00

20:0

10.332

9.133

3.2.3 The fatty acid composition obtained for the non-confluent sample.

The sum of the total area mV* (milli volt) minute is 34.78. Refer to appendix for calculations.

Retention time (minutes)

Carbon chain length of fatty acid

Area mV* min

Percentage of fatty acid composition (%)

11.40

16:0

8.420

24.21

11.55

16:1

2.130

6.12

14.16

18:0

7.275

20.91

14.41

18:1

2.496

7.18

16.19

18:2

10.575

30.4

16.73

20:0

3.884

11.17

From Table 3.2.2 it can be seen that the area under each peak from the GC was calculated and thus the fatty acid composition was figured out. In this sample there is a greater percentage fatty acid composition for linoleic (18:2) and stearic acid (18:0) which is 26.77% and 32.21%. This is followed by palmitic acid (16:0) that is 18.41%. Also at retention time of 17 minutes a peak was produced that is stated to be Arachidic acid (20:0). This is a saturated fatty acid.

On the other hand, from Table 3.2.3 it can be seen that there is a greater fatty acid composition for linoleic acid (18:2) and palmitic acid (16:0) which 30.4% and 24.21%. This is followed by stearic acid (18:0). The fatty acid, Palmitoleic acid that has a chain length of 16:1 is only present in the non-confluent sample.

Combine the above 2 tables so then a comparison can be made

Figure 3.3.1: The separation of phospholipid components.

Key

PI

Phosphatidylinositol

PS

Phosphatidylserine

PG

Phosphatidylglycerol

PE

Phosphatidylethnolamine

PC

Phosphatidylcholine

C

Cholesterol

OL

Other lipids

P14 C

Confluent

P14 NC

Non- confluent

Table 3.3.1 The Rf values obtained of the phospholipids standards

Standards

RF values obtained

Phosphoatidylcholine (PC)

0.121

Phosphatidylglycerol (PG)

2.837

­­Phosphatidylinositol (PI)

0.042

Phosphatidylethanolamine (PE)

0.369

Phosphatidylserine (PS)

0.709

Table 3.3.2 The lipids present in the P14 confluent, non-confluent samples and the Rf values

P14 confluent lipid sample

P14 non-confluent lipid sample

Lipids present

Rf values

Lipids present

Rf values

Phosphotidylcholine

0.132

Phosphotidylcholine

0.129

Phosphotidylserine

0.769

Phosphotidylserine

0.752

Cholesterol

0.751

Cholesterol

0.773

----

----

Other lipids

0.829

4 DISCUSSION

The aim of the experiments was to quantify and characterise lipid components potentially involved in the regulation of the proliferation of human glioma cell lines. Techniques such as lipid extraction, thin layer chromatography, gas chromatography and enzymatic assay were used to separate phospholipids, fatty acids and cholesterol.

Results obtained from the cholesterol assay showed a decrease in cholesterol concentration in confluent cells compared to non-confluent cells. For gas chromatography fatty acid methyl esters eluted from the samples were identified by looking at the retention times from the standards. Pentadecanoic was used as an internal standard and to allow quantification. Also linoleic and stearic acid were used to determine the retention times of key fatty acids so therefore the fatty acid profile could be figured. There is a greater fatty acid composition of fatty acid methyl esters in confluent cells compared to the non-confluent.

Is fatty acid used up or not?

A thin layer chromatography experiment was conducted to analyse which types of phospholipids were present in 1321n1 confluent and non confluent cells. Phosphatidylcholine and phosphatidylserine were present in both confluent and non-confluent tumour cells. It was observed that sphingomyelin was not identified in either of the glioma cells.

It is stated that the human tissue of the brain has a high amount of lipid content. However in a glioma which is a primary brain tumour, lipid content begins to decrease (Kohler, Machill, Salzer & Krafft, 2009). A brain tumour is a condition where there is an abnormal growth of cells.

The human glioma cell line 1321n1 is from an astrocytoma tumour that is found in the brain (Sigma-Aldrich, 2010). It is found to be very common in adults. The passages 13 and 14 have undergone the cell cycle 13 or 14 times. Non confluent is defined as rapidly dividing tumour cells where as confluent means that the cells are not dividing (REF).

4.1 Cholesterol Assay

The cholesterol oxidase oxidised the free cholesterol and produced cholest- 4- en-3- one as well as hydrogen peroxide, which oxidatively coupled with 4- aminophenazone and phenol. This produced a quinoneimine dye (Allain, 1974), which was pink in colour, and was measured at wavelength of 492nm. As the cholesterol concentration increased, the absorbance value also increased. This was because as there was more free cholesterol more cholest- 4- en - 3- one as well as H2O2 was produced which meant more cholest- 4- en - 3- one was able to oxidatively couple with 4- aminophenazone and phenol. This produced a higher intensity quinoneimine dye, (more pink in colour) which resulted in a higher absorbance value being measured.

The assay was measured after 1 hour, 2 hours and 3 hours of incubation at room temperature. This was done to optimise the assay. After 1 hour of incubation the assay had not developed fully. The optimisation of the assay was at 3 hours of incubation. If left any longer then the cholesterol at the lower concentration would have had absorbance values that increased more and there would be no differentiation between the absorbance values of the lower and higher cholesterol concentrations.

There was a difference between the absorbance values measured at the same concentration. There was a difference of 0.079 between the hours of 1 and 2 at the cholesterol concentration of 15 mmol/l. At the same concentration, there was less of an increase between the hours of 2 and 3, where the difference was 0.039. This showed that the optimisation of the assay was being reached.

The confluent 1321n1 P13 and P14 cell line showed a decrease in the free cholesterol concentration compared to the Non-confluent cells. This would be more precise if the total number of cells was known. If the total number of cells was known then a student T- test would be carried out to see if there was a significant difference between the confluent and non-confluent cells.

There is evidence to suggest that confluent cells should contain higher levels of lipid rafts compared to non- confluent (Chung, 2006). This would supply the tumour cell with cholesterol which is involved in proliferation of tumour cells. This explains why less cholesterol was seen in confluent lipid sample compared to the non- confluent lipid sample.

4.2 Preparation and extraction of FAMES for GC analysis

Many studies have been conducted to see the effects of polyunsaturated fatty acids on gliomas for example gamma linolenic acid and linoleic acid (Tettamanti, Goracci & Lajtha, 2009). It is stated that polyunsaturated fatty acids cause apoptosis of a tumour cell. This also includes glioma cell lines.

Research that has been conducted in the past states that fatty acids in the brain provides energy within all cells. An increase of fatty acid synthesis and use, contributes to the growth of a brain tumour cell (Stimson, 2010).

For FAME analysis a gas chromatography (GC) was used. The oven was maintained at a temperature of 100oC and was temperature programmed to 240oC at 12oC every 1 minute. The injector temperature was 50oC and detector was 250oC respectively. Before injecting 2µl of the sample into the GC, petroleum ether was added all around the sides of the conical glass centrifuge tube using a GC syringe. This was so then all the lipids present would be absorbed. Each sample was run for a total time of 40 minutes.

‘The time taken for a solute to transverse the whole length of the column is defined as the retention time' (Tranchant, 1969). The partition coefficient becomes changed due to the effect temperature has on a gas chromatography retention time (Baugh, 1993). Retention time is reported in minutes.

Standards

The results from the gas chromatography showed that for the standards a solvent peak was produced at a retention time of 2 minutes.When this is shown, indication is given that the sample is running effectively. At a time of 13.38 minutes a peak was identified to be pentadecanoic acid methyl ester (15:0), 16.70 minutes was linoleic acid methyl ester (18:2) and at 15.85 stearic acid methyl ester (18:0). The GC oven was left to cool to a temperature of 50oC before the next sample was injected.

However when conducting the experiment for the 1321n1 P13 and P14 confluent and non confluent samples results were not as accurate and efficient as expected. It was soon realised that this was due to distilled water being added to the methylating solution. If the methylating solution is not properly prepared then this can definitely affect the outcome for the results from the gas chromatograph.

Confluent

This was corrected and firstly the standards were run again followed by the sample which consisted of the 1321n1 P14 and P13 confluent cells. From the GC the fatty acid methyl esters, palmitic (16:0), linoleic (18:2) and stearic (18:0) were identified at retention times of 11.67, 14.45 and 16.49 minutes.

There also were many other fatty acids present in the confluent sample than seen in the non confluent sample. The peaks indentified were polyunsaturated and saturated fatty acids. “Saturated fatty acids are synthesised from acetyl-CoA and malonyl-CoA to palmitic acid and then elongated to longer chain fatty acids” (Meng, Riordan, Mikirova et al 2004).

A peak produced at a retention time of 9.60 minutes showed to be an anomalous result. This could be due to that there is a detergent present or that the tube was not washed out properly.

Non confluent

On the other hand for the non confluent sample all three peaks were produced to similar retention times of the standard fatty acid methyl esters but it does show that the fatty acid composition does differ in a confluent sample in comparison to a non- confluent human glioma cell line. This is because there is a greater fatty acid composition of linoleic acid and stearic acid in the confluent sample. This is due to an increase in proliferating tumour cells which indicates a greater amount of lipid rafts present. However for palmitic acid the percentage for the fatty acid composition is greater in the non- confluent sample.

Sphingomyelin

Sphingomyelin is any of the sphingolipids which yields sphingosine, choline, a saturated fatty acid and a phosphoric acid upon hydrolysis. It is found in primary nervous tissues (Anon, 2010).

“Sphingomyelin is a lipid that consists mostly of fat content in the cell membrane”. The basic structure of sphingomyelin is a polar group consisted of a phosphoethanolamine or phosphocholine, a fatty acid which is saturated and a sphingosine (REF).

When sphingomyelin is synthesised diacylglycerol is produced. This plays an important part in protein kinase C activation and is also involved in cell proliferation (Cerbon, 2009).

For the sample analysis report of sphingomyelin linoleic and stearic acid were present in the sample. After calculating the fatty acid composition for each it was concluded that there is a greater percentage of linoleic acid.

This time when conducting the experiment there were a lot more lipids present in the sample compared to previous times. It explains as to why results were more accurate and consistent for the confluent, non confluent and sphingomyelin.

4.3 Statistical Analysis

An independent t-test would be carried out between the confluent and the non confluent samples to see if the results were significantly different. Studies have shown in the past that the cholesterol concentration decreases in tumour cells.

A statistical was performed to show the significant difference in fatty acid composition between the confluent and the non confluent P13 and P14 cell lines. The type of test that was considered is a student's t - test.From previous studies a significant difference between the confluent and non confluent would show as a P value greater than 0.05. However a P value less than 0.05 would be considered as insignificant (P<0.05).

Furthermore a ratio between the cholesterol concentration and fatty acids would be determined for the tumour cells. This is to check which types of lipids are in greater quantity in the tumour which would contribute to the proliferation of human glioma cell lines.

4.4Thin layer chromatography

The result obtained from the TLC plate, showed that clearly that the phospholipids phosphatidylserine and phosphatidylcholine were present in the lipid sample. In comparison, the confluent sample had a higher intensity of phosphatidylcholine than the non-confluent. There is significance in this statement as past research shows more proliferation in phosphatidylcholine than any other phospholipid. In addition to this cholesterol was also identified. As some phospholipids are polar and some are non-polar, it plays an essential role in why each sample moves differently along the plate. As cell membranes are made up of phospholipids, the fluidity varies in the polar and non-polar regions thus the phospholipids have different density. Figure 3.3 shows how far each phospholipid travelled up the TLC plate.

Past research states malignant tumours contain larger amounts of phospholipids and cholesterol than in benign tumours (Haven, 1936). But it also stated that fat is a source of energy for the rapid growth of tumours i.e cholesterol (Haven, 1936). Due to the identification of nuclear specific phosphatidylcholine, biosynthetic and signalling pathways have given credibility to the hypothesis that specific molecular species of phosphatidylcholine are involved in cell proliferation.

Phosphatidylcholine is hydrolysed by phospholipase D to generate phosphatidic acid. Rapamycin is a drug that helps to stop the body from rejecting bone marrow or organ transplants. It does this by blocking specific white blood cells that attack foreign cells. Rapamycin (also referred to as Sirolimus) is a ‘type of antibiotic, immunosuppressant, and a type of serine/threonine kinase inhibitor' (National Institute of Cancer).There are two component complexes of mammalian target of rapamycin which are C1 and C2. The mammalian targets of rapamycin mediated signals that promote cancer cell survival are regulated by phosphatidic acid (Foster, 2009). GTPase such as Ras gene is also regulated by phosphatidic acid which is involved in kinase signalling pathways that controls transcription of genes which regulate cell growth and differentiation.

Research shows that at least two classes of phospholipids are present in tumours (Haven, 1936). This is significant as the results obtained showed that two types of phospholipids were present and a substantial amount of cholesterol was also present (Haven, 1036).

There are advantages of the cholesterol assay such as it is inexpensive and easy to replicate. There is also no need for qualified personnel to carry out the assay. The enzymatic cholesterol assay can give an accurate reading of the absorbance values if performed correctly.

The advantages of the gas chromatography technique are that no extra equipment is required for a measurement to be produced for example the retention time. This saves on time and money. It also produces a fast analysis within minutes, a high resolution is produced so is efficient, very sensitive, quantitative analysis, small samples are required for example in µl and is reliable (McNair & Miller, 2009). In relation to the GC column, ‘It is stated that column efficiency increases with column length' (McNair & Miller, 2009). On the other hand, in scientific institutions and industrial organisations, gas chromatography has become a routine when wanting to characterise fatty acid profiles of lipids in biological materials (Liu, 1994). In addition it is also widely used for quantitative and qualitative analysis mixtures, the purification of compounds, the determination of thermo chemical constants as heats of solution and vaporisation, vapour pressure and activity coefficients (Linde, 2008).

TLC has many different advantages in obtaining good results in a short span of time with very little cost efficiency. TLC enables visual results and is the only chromatographic method which offers results as an image in either fluorescence or a dark coloured form on the zones of the plate (Reich & Schibli, 2007). This is a major advantage as a visual image has high significance as to what material have moved up the plate and can easily be marked or a picture may be taken to analyze the results and can easily be kept for future reference. In comparison to HPLC which produce long peak tables, TLC results are easier to communicate (Reich & Schibli, 2007). As TLC has its own simplicity, it also has the advantage that HPLC does not have, parallel analysis of samples. It is a very easy and simple method of plotting both the standards and samples on one plate therefore making analysis a very simple step, whereas HPLC all samples have to be run individually therefore more time consuming. However HPLC is a far more expensive procedure and just running the machine and waiting for the samples to be inserted mounts up the costs. As flexibility is a concern, TLC over rules HPLC. This is due to the parameters that TLC establishes. Experiments can be carried out without time difficulties as TLC does not require a start up time and therefore results are presented rapidly.

5 CONCLUSION

In conclusion it can be said that the free cholesterol concentration decreases in 1321n1 confluent glioma cells. Linoleic and stearic acid methyl esters increased in fatty acid composition in confluent glioma cells; however the palmitic acid methyl ester decreased. Phophatidylcholine (PC) and phosphatidlyserine (PS) are both present in confluent and non-confluent cells when compared to known standards.

6 LIMITATIONS

However when conducting the experiment there were weaknesses present in some of the materials used and the way the protocol was conducted. A problem in the materials was that at times the glass and gilson pipettes were not precise in measurement as there was a pipetting error before transferring the samples into the methylating tubes and also for the cholesterol assay as the samples were pipetted into the microtitre plate. This could be because a 1ml gilson is not calibrated which could affect the outcome of results. In addition a technical error occurred with the nitrogen gas cylinder so therefore at times the petroleum ether was not dried out fully in the conical glass centrifuge tube. This could explain why there was an anomalous result in the confluent sample. As only 2µl of sample was injected into the GC it is always beneficial to make sure there is no air present in the GC syringe as this could affect the concentration of lipids and the outcome of results from the gas chromatography. Thus it is always essential to draw up more of the sample than required. Further more if any problem did occur during analysis then a new sample was needed (Latorre, Rigol, Lacorte & Barcelo, 2003).

For the cholesterol assay, when pipetting the samples in the microtitre plate, they were sometimes on the sides of wells and not in the middle wells. This is a limitation as some absorbance values may be measured incorrectly; as a result of this an inaccurate concentration would be measured.

On the other hand,

The lid of the glass tank was not correct or suitable which resulted in no results for the first couple of weeks. This got replaced.

7 SCOPE FOR FURTHER STUDIES

Free cholesterol decreases in brain tumours, but it is said that cholesterol esters increase in brain tumours. This can be determined using the technique of Raman spectroscopy and mass spectrometry (Kohler, 2009). It has a higher sensitivity than the enzymatic cholesterol as that was carried out which will enable the cholesterol ester: cholesterol ratio to be carried out (Kohler, 2009). The cholesterol assay was a colorimetric one. There is another technique that offers a fluorometric assay; this is around 4-10 times more sensitive than the colorimetric assay (Anon (a), 2009). This should allow an increase in the validity of the results.

The gas chromatography technique is a sufficient test for fatty acid methyl ester analysis, a more accurate and precise method is the high performance liquid chromatography (HPLC) technique. This could be used for future research to gain sufficient results about the lipid components potentially involved in the regulation and the proliferation of human glioma cell lines. The major difference between HPLC and GC is that a GC can only separate substances that are volatile or at elevated temperatures can be evaporated intact and easily obtains volatile derivatives (Meyer, 2004). An advantage of HPLC is that the mobile phase has a higher viscosity in comparison to a GC. This is important because it results in a smaller diffusion coefficient and resistance of the mobile phase is high flow. In the mobile phase the flow velocity is constant throughout the whole length of the column (Meyer, 2004).

Liquid chromatography mass spectrometry is a better technique than gas chromatography and is time efficient. With this method, identification of phospholipids such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC) and sphingomyelin was obtained. The combination of molecular species produced were PE (C18:0/C20:4), PG (C16:0/C18:1), PI (C18:0/C20:4), PC (C18:0/C18:0) and sphingomyelin (d18:1/C18:0) (Pang, Liang, Wang et al 2008).

Factors that could be investigated for future research in this area are to identify a peak using additional investigations such as TLC mobility, column fractionation (Leray, n.d.). In addition the protein content in a human glioma cell line can also be extracted and analysed with its effects in regards to proliferation for example a protein called the epidermal growth factor (EGF) receptor (Stimson, 2010).

HPLC is a more progressive and better technique but it requires a high amount of cost to run, as a specialist is required to run the program. LMCS is another technique that can be used in future studies, as it will be efficient if using the same lipids. This is because LMCS has the ability to measure both fatty lipids and phospholipids at the same time therefore a difference can be pointed out immediately.

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