CHAPTER 3

LIPID CHEMISTRY

GROUP 4 SEC. A1

BALANSAG, JOANNEBALBASTRO, ROWENABALAORO, FRANCEBANTUAS, ALMAIRABARCELONA, LULUBELLEBARLOLONG, ROLDAN

PROCTOR:NICO D. CALINGACION, M.D.

EXPERIMENT 1: EXTRACTION, FRACTIONIZATION AND IDENTIFICATIONOF BRAIN LIPIDS

The brain has a high content of lipid compared to all other tissues of the body. It contains a variety of lipids. Complex lipids such as the phosphatides, cerebrosides, and sphingosides contain fatty acid components of the C-16 to C-24 carbon chains, chiefly in the form of stearic, oleic, linoleic and arachidonic acids are found in the brain. The steroids alcohol, cholesterol, occurs abundantly in the brain.

This experiment may be divided into four major parts:1. Extraction of the total lipids.2. Isolation of the triglycerides and saponificaiton of the triglycerides make way for

the study of soaps, fatty acid and glycerol.3. Isolation and tests on lecithin4. isolation and tests on cholesterol

Objectives:1. To be able to study the properties of various lipids.2. To be able to classify and identify the lipids.

Procedure:30 grams of pig’s brain

(homogenized in ether-alcohol solution)TOTAL LIPID EXTRACT

2/3 portion 1/3 portion Isolation of Triglycerides Lecithin-Cholesterol Isolation

20 ml portion 30 ml portionTest on Soaps A. Test on Fatty Acids A. Test on Lecithin1. Salting Out 1. Solubility 1. Acrolein 2. Insoluble Salts 2. Translucent Spot 2. Iodine Absorption3. Surface Tension 3. Iodine Absorption 3. Phosphorus

B. Test on Glycerol B. Test on Cholesterol 1. Solubility 1. Liebermann- 2. Translucent Spot Burchard Test 3. Acrolein 2. Salkowski Test 4. Benedict’s Test

20 ml PortionTest on Soap

Salting Out Insoluble Soaps Surface Tension 5 ml of 5 ml 5 ml 5 ml 5 ml Soap 5 ml Solution Soap Solution Distilled Water Solution 2% NaCl

Add Add Add Add NaCl CaCl2 MgSO4 Sulfur Powder

Precipitate Precipitate Precipitate

Add Add Add Observe Water Water Water

Observe Observe Observe

Results and Observations

Test Obtained Results Expected ResultsSalting Out A white precipitate was

formed, and dissolved upon addition of water.

White precipitate which dissolves in water.

Insoluble salts CaCl2 and MgSO4 formed white precipitates and upon contact with water, it did not dissolve.

White precipitates which are insoluble in water

Surface Tension In water: The sulfur powder stayed at the water surface (floated).

In soap solution: The sulfur powder went at the bottom of the tube (sank).

In 2% NaCl: The sulfur powder stayed at the water surface (floated).

In Water: The sulfur powder floats.

In soap solution: The sulfur powder sinks.

In 2% NaCl: The sulfur powder floats.

Discussion:

Lipids that contain fatty acid ester linkages can undergo hydrolysis, a reaction that is catalyzed by strong acid or base. Alkaline hydrolysis of fatty acid esters is known as saponification. Saponification of triglycerides produces glycerol and fatty acids salts. High molecular mass of fatty acid salts are commonly referred to as soap. The chemical reaction between a triglyceride and a base to form glycerol and fatty acids salts is shown below.

When the hydrolyzed solution is saturated with NaCl, the soap separates. This principle is

used in manufacturing soaps. Insoluble soaps are formed when calcium and magnesium react with the higher fatty acids of soaps and forms an insoluble white curd like precipitate of magnesium or calcium salts (scum).

Surface tension expresses the force with which the surface molecules attract each other. It is also a property of liquids arising from unbalanced molecular cohesive forces at or near the surface, as a result of which the surface tends to contract and has properties resembling those of a stretched elastic membrane. Water has a high surface tension because of its hydrogen bonding as shown below.

Substances that break hydrogen bonding of water that causes to lower its surface tension are called surfactants. Soap is an example of surfactant. Soaps have an unusual behavior in water because it comprises both hydrophilic and hydrophobic regions. The cleaning action of soaps is due to the arrangement of the hydrophilic (polar) heads, which are the carboxyl groups; and hydrophobic (nonpolar) tails are the hydrocarbon chains. When in water the surfactant molecules reversibly assemble into polymolecular aggregates called micelles. By gathering the

hydrophobic chains together in the center of the micelle, the grease or oil present in the solution will be emulsified by the hydrophobic tail inside of the micelles and it is rinsed away.

Hydrophilic heads

Hydrophobic tails

Hydrophobic Hydrophilic (nonpolar) tail (polar) head

30 ml PortionA. Test on Fatty Acids

Solubility Translucent Spot Test Iodine Absorption TestHeat the solution Small amount of precipitate Small amount of precipitate

Add HCl Ordinary writing paper Add 5 ml of CHCl3

Precipitate Observe Add Hubl’s iodine

Place in water Place in CHCl3 Observe

Observe

30 ml PortionB. Test on Glycerol

Solubility Acrolein Test Translucent Benedict’s Test Spot TestPrecipitate 0.5 grams of powdered Precipitate Litmus paper KHSO4

If acidic

Water CHCl3 Add 10 drops of Ordinary writing Add dilute Glycerol solution paper Na2CO3

Heat in 5 ml Benedict’s Reagent

Observe Note the odor Observe Add 5 drops of Glycerol solution

Boil for 2-3 mins.

Observe

Results and Observations:

FATTY ACIDTest Obtained Results Expected ResultsSolubility test

H2OCHCl3

insolublesoluble

insolublesoluble

Translucent Spot test It formed a spot and remained even after long standing

There is a presence of translucent spot and remains even after long standing

Iodine Absorption test The iodine solution was decolorized

The iodine solution decolorized

Discussion:

The degree of solubility in water and in nonpolar solvents of fatty acids will depend on whether it is a saturated or unsaturated and if it is saturated whether it is short-chain, medium-chain or long-chain fatty acids. Below is a table showing the solubility of a short chain saturated fatty acid, long-chain saturated fatty acids and unsaturated fatty acids in water

Short-chain saturated fatty

acids

Long-chain saturated fatty

acids

Unsaturated fatty acids

Solubility in Water Limited solubility Insoluble Insoluble

However, glycerol behaves differently in water from fatty acids. Glycerol is soluble in water but insoluble in nonpolar solvents. Glycerol is soluble in water because it contains three hydroxyl or alcohol groups. These hydroxyl or alcohol groups are hydrophilic and are responsible the solubility of glycerol in water.

GLYCEROLTest Obtained Results Expected ResultsSolubility test

H2OCHCl3

solubleinsoluble

solubleinsoluble

Translucent Spot test It formed a spot but it did not remain even after long standing

There is a presence of translucent spot and remains even after long standing

Acrolein Test pungent odor Pungent odorBenedicts test negative positive

Another test for fatty acids and glycerol is the translucent spot test, which is a simpler test for the lipids wherein marks on an unglazed paper shows the presence of triglycerides (fats/glycerol). Below is a figure of a translucent spot test. On the left is a negative result while on the right shows a spot which is a positive result.

NEGATIVE POSITIVE (No spot formed) (A translucent spot is formed)

The volatility of fatty acids will also depend whether the fatty acids are saturated or unsaturated and if saturated whether it is a short-chain, medium-chain or long-chain fatty acids. Below shows the difference of short-chain, long-chain fatty acid and unsaturated fatty acid.

Short-chain saturated fatty

acids

Long-chain saturated fatty

acids

Unsaturated fatty acids

Volatility Volatile Nonvolatile Nonvolatile

Acrolein test is a test for the presence of glycerin or fat. Acrolein (CH2=CH-CHO), is released if the test is positive. When a fat is heated strongly in the presence of a dehydrating agent such as KHSO4, the glycerol portion of the molecule is dehydrated to form the unsaturated aldehyde. This unsaturated aldehyde is produced by the loss of water by the glycerol portion of the fat molecule. In biological membrane this will also result in the formation of free radicals, a process known as lipid peroxidation.

Glycerol Acrolein (pungent odor)

Another reaction exhibit by fatty acids is halogenation reaction which is the basis of the chemical procedure known as Iodine Absorption test. It is use for determination of unsaturation of fats. Halogens ( I, Br ) will add across the double bonds and thus the decolorization of an iodine or bromine solution will indicate the presence of unsaturated fatty acids. Iodine from

Huble's solution adds up across the unsaturation sites of oil or fat giving a colourless product and a little more of iodine gives a permanent violet colour to the solution.

H3C(CH2)16COOH + I2 no reaction H H H H H3C(CH2)7 – C =C – (CH2)7COOH + I2 H3C(CH2)7—C – C – (CH2)7COOH I I

Benedict's test identifies reducing sugars based on their ability to reduce the cupric (Cu2+) ions to cuprous oxide at basic (high) pH. Cuprous oxide is green to reddish orange. In this carbohydrate test, a green solution indicates a small amount of reducing sugars, and reddish orange indicates an abundance of reducing sugars. Nonreducing sugars such as sucrose produce no change in color (the solution remains blue). Monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=0) groups that reduce weak oxidizing agents such as the copper in Benedict's reagent. Benedict's reagent contains cupric (copper) ion complexes with citrate in alkaline solution.

1/3 PortionA. Test on Lecithin

1/3 portion

Heat until syrupy consistency

Add 15 ml of ether

Stir

Add 15 ml acetone

Filter

Precipitate (lecithin) Filtrate (cholesterol)

Precipitate (lecithin)

Acrolein test Iodine Absorption Test Test for Phosphorus0.5 grams of powdered Small amount of Small amount of KHSO4 Precipitate Precipitate

Add 10 drops of glycerol Ordinary writing paper Add 5 ml of 10% HCl Solution

Heat Boil for 10 mins.

Note the odor Observe Add equal volume of Conc. HNO3 and ammonium molybdate solution

Warm

Yellow crystals

Filtrate (cholesterol)

Evaporate the ether-acetone filtrate

Cool

Add CHCl3

Filter

Clear solution

Salkowski test Liebermann-Burchard test

2 ml 2 ml

Add equal volume of H2SO4 Add 10 drops acetic anhydride and CHCL3

Observe Add 2 drops of conc. Sulfuric acid

Stand for 15 mins.

Note the end color

Results and Observations:

Lecithin

TEST Obtained Result Expected ResultAcrolein test It produces an unpleasant odor

(pungent)Pungent

Iodine absorption test Pinkish color of precipitate region upon dropping of

iodine(decolorization of iodine)

Decolorization of iodine

Test for phosphorus Yellow precipitate formed and formation of crystals upon

cooling

Yellow precipitate formed and formation of crystals upon

cooling

Cholesterol

Test Obtained result Expected result

Salkowski test CHCl3: cherry red colorAcid layer: clear

CHCl3: Cherry red colorAcid layer: fluorescent green

Liebermann-Burchard reaction

Dark green color Green color

Discussion:

As aforementioned, Acrolein test is a test for the presence of glycerine or fat. Lecithin or phosphatidyl choline is a phosphoglycerides which, the parent compound is phosphatidic acid and its structural backbone is glycerol. Lecithin produce acrolein and will yield expected result in iodine absorption test because the presence of double bonds. As we stated earlier that lecithin has a phosphatidic acid as it’s parent compound, it will yield yellow crystal, an expected result in test for phosphorus. Test for phosphorus detects the presence of free phosphate in acidic solution can be detected by adding a molybdate to the solution.

HPO42-(aq) + 12MoO4

2-(aq)+3NH4+(aq)+23H3O+(aq) (NH4)3[P(Mo3O10) 4](yellow,s) + 35 h20(l)

The equation above illustrates the pertinent reaction between phosphate and ammonium molybdate solution in presence of nitric acid.

When lipids contains phosphate groups in their structures are added to a strong acid solution such nitric acid, the lipid hydrolyses, producing free phosphate. The free phosphate then reacts as in equation above, forming yellow precipitate which forms crystals upon cooling.

For cholesterol analysis we perform the Salkowski test and the Liebermann-Burchard Test.

The Salkowski test is a qualitative analysis of cholesterol detection in natural products. Salkowski reaction is the reaction of cholesterol with concentrated sulphuric acid. Concentrated sulphuric acid is highly hygroscopic and it removes two molecules of water from two molecules of cholesterol, it causes a connection at position 3, forming bi- cholestadien (a). Simultaneously the sulphuric acid sulphonates the molecule of bi-cholestadien at positions 7,7’ of aromatic ring and, as a final product, red colour bi-sulphonic acid of bi-cholestadiene is formed (b).

a.

bi-cholestadien

b. bisulfonic acid of bi-cholestadiene

Liebermann-Burchard Test on the other hand is a colourimetric test for unsaturated sterols, notably cholesterol; a blue-green color develops when such substances are added to acetic anhydride and sulphuric acid in chloroform

Some biological importance of lipids

Surfactant in lungs is dipalmitoyl lecithin which lowers the surface tension in the alveoli. The result of cohesive forces where molecules pulls each other inward is surface tension. This collapses the alveoli, but because there is a presence of surfactant in our lungs, it lowers the surface tension thus preventing it to collapse during expiration also a study is conducted that surfactant proteins present in the lungs can recognize bacterial, viral and fungal surface oligosaccharides and thus can opsonize these pathogen

Bile Acids1. Primary Bile Acids

Cholic Acid and chenodeoxycholic AcidSynthesized in the liver from cholesterol

2. Secondary Bile acidsDeoxycholic acid and lithocholic AcidSynthesized in the small intestines from the primary bile acids

FUNCTIONS:Aid in fat digestion and absorptionFacilitate absorption of fat-soluble vitamins (A,D,E,and K)Help in solubilization of cholesterol in bile

C21H30O2 Progesterone

Chemical name:Preg-4-ene-3,20-dione FUNCTION Intermediate metabolite in biosynthesis of other steroid hormones Maintain pregnancy during the first trimester Involved in menstrual cycle Drug for threatened abortion Component of oral contraceptives

C21H30O5CortisolChemical name: 11beta,17alpha,21-Trihydroxypegn-4-ene-3,20-dioneFUNCTIONSRegulate intermediary metabolismImmunosuppressive and anti-inflammatory actionPlay an important role in stress response.

C21H28O5Aldosterone

FUNCTIONS Regulates water and sodium metabolism Involved in blood pressure regulation

C19H28O2Testosterone

Chemical name:17beta-Hydroxyandrost-4-en-3oneFUNCTIONSNormal male sexual development of the embryoDevelops and maintain male secondary sexual characteristicsImportant for male fertility

C18H24O2 Estradiol

Chemical name: estra-1,3,5(10)-triene-3,17beta-diol FUNCTIONS Normal female sexual development Development of female secondary sexual characteristics Induce differentiation of mammary glands Involved in menstrual cycle Affects synthesis of neurotransmitter and receptor protein in the CNS Influences lipid metabolism – increase HDL, decrease LDL