Lipids - academic.mu.eduLIPIDS Carbohydrates are defined in terms of the organic functional groups...

LIPIDS

Carbohydrates are defined in terms of the organic functional groups present on the molecule. They are polyhydroxyl aldehydes or ketones. Carbohydrates are very polar molecules because there is a large difference in electronegativity between oxygen and carbon and between oxygen and hydrogen. They are very water soluble because the hydroxyl groups and carbonyl groups can form numerous hydrogen bonds with water.

LIPIDS are not defined in terms of the functional groups present on the molecule, rather they are defined on the basis of their physical properties. Lipids are the non-polar, hydrophobic, water insoluble components of the cell. Lipids are soluble in non-polar solvents such as ether, benzene, the liquid alkanes, or the halogenated alkanes. They cannot form dipole-dipole interactions nor hydrogen bonds with water. They interact with each other and the non-polar solvents by very weak London forces or hydrophobic interactions.

Lipids are a diverse group of biological molecules. There are numerous different classes of lipids each with slightly different chemical, physical, and biological properties.

Saponification

When animal fat (lipids) are treated with a strongly basic solution (50% solution of NaOH or KOH) and heat, some of the lipids leave the oily mass of animal fat floating on the surface and enter the water phase. The hot NaOH has catalyzed the hydrolysis of some of the lipids in the sample into smaller more water soluble molecules. Hydrolysis {hydro - water; lysis - to break} is the breaking of chemical bonds by adding water across them.

Question - What types of chemical bonds, what organic functional groups, are hydrolyzed by hot strongly basic solutions; e.g., 50% NaOH, KOH or LiOH?

Hot sodium hydroxide catalyzes the hydrolysis of Ester Bonds, Amide Bonds, and some Glycosidic (Acetal or Ketal) Bonds. The base catalyzed hydrolysis of esters and amides is termed SAPONIFICATION and the group of lipids that are hydrolyzed into smaller more water soluble molecules by hot base are called the SAPONIFIABLE LIPIDS. Saponifiable lipids are large water insoluble molecules composed of smaller water soluble and/or slightly water soluble precursors covalently linked by ester, amide, and/or glycosidic bonds. Those lipids that did not react with the hot NaOH or KOH solution are called the NONSAPONIFIABLE LIPIDS, they remain floating on the top of the pot.

The “Small Molecules” Produced by the Saponification of Lipids or

The Precursors of the Saponifiable Lipids

The Fatty Acids

The largest group of molecules produced by lipid saponification are the Fatty Acids. Fatty acids are long chain monocarboxylic acids containing a minimum of eight carbon atoms. Eight carbons is the dividing line with respect to solubility; carboxylic acids with less than 8 carbon atoms are relatively water soluble, those

©Kevin R. Siebenlist, 20201

with 8 or more are only slightly water soluble. Some of the fatty acids are saturated, containing only carbon-carbon single bonds and a maximum amount of hydrogen. Others are unsaturated, containing one or more carbon-carbon double bonds. Fifteen (15) fatty acids make up the majority (≈65%) of the fatty acids found in human cells. These 15 common fatty acids are:

Nine of the 15 common fatty acids are saturated fatty acids, the other six fatty acids are unsaturated. Palmitoleate & Oleate are monounsaturated fatty acids since they contain one carbon-carbon double bond. Linoleate, α-Linolenate, γ-Linolenate, & Arachidonate are polyunsaturated fatty acids, they contain two or more double bonds. {Note: The structure of the common fatty acids are given in Lipids Handout. It is necessary to be able to identify the structure of these molecules.}

Things to note about the common fatty acids:

Name when Unionized

Name when Ionized # C’s

# double bonds Location of Double Bonds

Shorthand Notation

Capric acid Caprate 10 0 — 10:0

Lauric acid Laurate 12 0 — 12:0

Myristic acid Myristate 14 0 — 14:0

Palmitic acid Palmitate 16 0 — 16:0

Stearic acid Stearate 18 0 — 18:0

Arachidic acid Arachidate 20 0 — 20:0

Behenic Acid Behenate 22 0 — 22:0

Lignoceric acid Lignocerate 24 0 — 24:0

Cerotic Acid Cerotate 26 0 — 26:0

Palmitoleic acid Palmitoleate 16 1 between 9 & 10 16:1 Δ9

Oleic acid Oleate 18 1 between 9 & 10 18:1 Δ9

Linoleic acid Linoleate 18 2 between 9 & 10 and 12 & 13 18:2 Δ9,12

α-Linolenic acid α-Linolenate 18 3 between 9 & 10, 12 & 13 and 15 & 16

18:3 Δ9,12,15

γ-Linolenic acid γ-Linolenate 18 3 between 6 & 7, 9 & 10 and 12 & 13

18:3 Δ6,9,12

Arachidonic acid Arachidonate 20 4 between 5 & 6, 8 & 9, 11 & 12, and 14 & 15

20:4 Δ5,8,11,14


1. They all contain an even number of carbon atoms. 2. At physiological pH, pH 7.4, the carboxylic acid group of the fatty acid is ionized. They have

donated a hydrogen ion to water or some other proton acceptor forming a negative carboxylate ion. The charged forms are named with the “ate” suffix used for carboxylate ions. Since the charged forms are the only form present at physiological pH the fatty acids will always be named as ions. The ionized forms are the ones given on the Lipids Handout.

3. The double bonds in the common unsaturated fatty acids are all in the “cis” configuration. The “cis” double bond puts a “kink” in the molecule.

4. The unsaturated fatty acids have lower melting points and boiling points that the saturated fatty acids. The “kink” in the unsaturated fatty acids makes it more difficult for these molecules to pack in an orderly array necessary for forming a solid. Likewise, the kinks weaken the interactions in the liquid phase making it easier for the molecules to enter the gaseous phase.

5. Two fatty acids; Linoleate and α-Linolenate; are essential fatty acids. These fatty acids cannot be synthesize by the human animal but they are necessary for its normal growth, development, and maintenance. They must be obtained from the diet either from plants that can synthesize them or from other animals that have eaten plants. {Note: γ-Linolenate can be synthesized from Linoleate.}

6. The sodium or potassium salts of the fatty acids, the products of saponification, are the soaps (saponification = soap forming).

“Trans Fats” have been deemed unfit for human consumption. “Trans Fats” are formed when the double bonds of unsaturated fatty acids in polyunsaturated triacylglycerols are reduced with a less than stoichiometric amount of H2 (partially hydrogenated). The conditions of this industrial reaction reduces some of the double bonds in the unsaturated fatty acids to single bonds. However, since there is a limiting amount of H2 some of the double bonds, rather than undergoing reduction, undergo a change in configuration from cis to trans. Note: these fatty acids are the trans isomers of the naturally occurring cis forms.

Not all fatty acids with trans double bonds are harmful to human health. A variety of naturally occurring fatty acids containing trans double bonds have been identified and have been shown to be beneficial for human health. These naturally occurring “good” trans fatty acids make up only small percentage of the fatty acids, they are all polyunsaturated, and they contain a mixture of “cis” and “trans” double bonds. These are in the Lipids Handout Document.

Question - How do soaps work? When fats are saponified the fatty acids are converted into their sodium or potassium salts. The carboxylate ions are weakly AMPHIPATHIC. Molecules that are AMPHIPATHIC contain a polar region, the charged carboxylate group and a non-polar region, the hydrocarbon “tail” of the fatty acid. When soaps are placed in water they coalesce to form spherical structures called MICELLES. The surface of the micelle is


composed of the charged carboxylate ions of the soap molecules. These charged groups interact with the polar water. The interior of the spherical micelle particle contains the non-polar hydrocarbon “tails” of the soap molecules. These non-polar hydrocarbon tails interact with each other by hydrophobic interactions and avoid water as much as possible. Dirt and odors are held to your body and clothing by bodily oils (lipid molecules) that we naturally secrete to maintain skin integrity. The hydrocarbon tails of the soap molecules dissolves the body oil into the micelle releasing the dirt (remember like dissolves like). The polar head of the soap allows it to interact with water and the water can wash the oils and dirt away.

Polar Alcohols

The second most abundant molecule in the products of saponification mixture, in the group of precursors of the saponifiable lipids, is the molecule glycerol. Glycerol is a three carbon tri-alcohol, hence it is very polar. Its IUPAC name is 1,2,3-propanetriol. Glycerol is used commercially as a skin softener and as a laxative.

Also present in the products of saponification, albeit to a lesser extent, are several other very polar alcohols. These polar alcohols include: ethanolamine, choline, serine, and inositol. Ethanolamine contains a polar alcohol group as well as a polar primary amine group. Choline contains a polar alcohol group as well as a positively charged quaternary ammonium ion. Serine contains a polar alcohol group, a primary amine group, and a carboxylic acid group. Serine is an amino acid. Inositol is a cyclic six carbon hexa-alcohol, it has six hydroxyl groups. Although it may be confused with a monosaccharide, inositol is not a sugar.

Ethanolamine, choline, and serine are charged molecules at physiological pH, pH 7.4. At this pH the amino groups on ethanolamine, choline, and serine accept a proton to become positively charged ammonium ions. The carboxylic acid group on serine will also ionize, it will donate a proton to become a negatively charged carboxylate ion. The structures of these small molecules are given on the Lipids Handout Document. The structures of ethanolamine, choline, and serine are given in the ionized forms, since only the ionized form exists in the cell. {It is necessary to be able to identify the structures of these small molecules that act as precursors for the saponifiable lipids.}

Long chain alcohols are also precursors for some of the saponifiable lipids. These alcohols all have the hydroxyl group on carbon 1 and can contain from 16 to 34 carbon atoms. Some of the alcohols found in lipids are vinyl alcohols. A vinyl alcohol contains the hydroxyl group on carbon 1 and a carbon-carbon double bond between carbon 1 and carbon 2.

Sphingosine

Sphingosine is an eighteen carbon molecule. There are polar hydroxyl groups on carbons 1 and 3 and a polar amino group on carbon 2. A carbon-carbon double bond in the “trans” configuration is present between carbon 4 and 5. The remainder of the molecule is hydrocarbon. Sphingosine is an AMPHIPATHIC molecule. Amphipathic molecules have a very polar region and a very non-polar region. The first three carbons of sphingosine contain three very polar groups, two hydroxyl groups and the positively charged amino group. The last 15 carbons are very non-polar, they are hydrophobic hydrocarbons. {Note: The structure of sphingosine is given in the Lipids Handout Document. It is necessary to be able to identify the structure of this molecule.}


Monosaccharides

Monosaccharides are present in the mixture of compounds produced when saponifiable lipids are treated with hot NaOH. The monosaccharides found most often are glucose, galactose, and mannose. Modified monosaccharides such as glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid (sialic acid), are also found in the products of saponification.

Last but not least, the PHOSPHATE ION (PO4–3) is found in the saponification mixture.

The Precursors of the Saponifiable Lipids - Summary (The Small Molecules that Make-Up the Saponifiable Lipids)

•Fatty Acids •Saturated & Unsaturated

•Polar Alcohols •Glycerol, Serine, Ethanolamine, Choline, & Inositol

•Sphingosine •Long Chain Alcohols

•Saturated and Vinyl •Monosaccharides & Modified Monosaccharides

The Saponifiable Lipids

Up to this point;

1. Lipids have been defined as the non-polar, water insoluble components of the cell. 2. Lipids have been classified as either saponifiable or nonsaponifiable. 3. Saponifiable lipids are composed of smaller molecules covalently linked by ester, amide, or

glycosidic bonds. 4. The small molecules that make up the saponifiable lipids have been examined (see above). 5. Nonsaponifiable lipids do not react with hot NaOH solutions implying that this group does not

contain chemical bonds that can be hydrolyzed under basic conditions.

It is now time to take the small molecules that are the precursors of the saponifiable lipids and put them together in a variety of ways in order to assemble the various types of saponifiable lipids. The saponifiable lipids are broken down into classes and subclasses, the precursor small molecules that they are composed of will be described, and then the structure and function of each of the intact lipids will be examined. As the structure of the classes and subclasses of saponifiable lipids are described, contemplate the small precursor molecules that are used to build the complex molecule and look for / focus on the one or two unique characteristic(s) that distinguish one class of lipid molecules from another.

There are three major classes of saponifiable lipids; the WAXES, the GLYCEROL LIPIDS, and the SPHINGOLIPIDS. As their names imply, the glycerol lipids all contain at least one molecule of glycerol and the sphingolipids all contain at least one molecule of sphingosine. Within the class of glycerol lipids, there are three subclasses; the TRIACYLGLYCEROLS, the PHOSPHOGLYCERIDES, and the ETHER LIPIDS. A subclass


of the ETHER LIPIDS are the PLASMALOGENS. The name Triacylglycerol describes the makeup of these molecules; there are three acids (triacyl) and a glycerol present. The name Phosphoglyceride describes the molecule as containing a phosphate and a glycerol. Other groups are present, but the subclass name doesn’t indicated what other groups. Ether Lipid indicates that this subclass contains an ether functional group. Again other groups are present, but the name doesn’t indicate what groups. The sphingolipid class contains two subclasses; the SPHINGOMYELINS and the GLYCOSPHINGOLIPIDS. Sphingomyelin denotes that this subclass contains sphingosine and it bespeaks to the source of first isolation; the Myelin of the nervous system. “Glycos” is a prefix meaning sweet or sugar, hence the glycosphingolipids contain a sugar or sugars as part of their structures. The glycosphingolipids are further divided into the CEREBROSIDES and the GANGLIOSIDES. These terms, Cerebroside and Ganglioside, indicates from where they were first isolated, the nervous tissue.

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Waxes

Waxes are the simplest group of saponifiable lipids. Structurally, waxes are long chain alcohols 16 to 34 carbons in length in ester linkage with long chain fatty acids 14 to 36 carbons long. The ester linkage joining the long chain acid and alcohol makes a wax saponifiable. Waxes function primarily as water proofing agents. Plants use waxes to prevent the loss of water from leaves, seeds, stems, and fruit. Bees use wax to prevent their honey from drying out and crystallizing. Aquatic and marine animals and birds use waxes to prevent the “wetting” of their fur and feathers. Whale oil is primarily a wax. Humans produce and secrete a wax in SEBUM, an oily substance secreted by the sebaceous glands that lubricates the hair and helps to keep skin moist and supple.

Triacylglycerols

Moving up in complexity the next class of lipids is the Triacylglycerols. Triacylglycerols are composed of three fatty acids (triacyl) in ester linkage to one molecule of glycerol. The fatty acids attached to glycerol can be any combination of the 15 fatty acids previously described as well as any of the “minor” fatty acids; fatty acids shorter, longer or the “good” trans fatty acids. A SATURATED TRIACYLGLYCEROL (SATURATED FAT) is formed when three saturated fatty acids are esterified to glycerol. Saturated fats are greasy solids at room temperature. A MONOUNSATURATED TRIACYLGLYCEROL (MONOUNSATURATED FAT) contains one unsaturated fatty acid and two saturated fatty acids. The number of double bonds in the unsaturated fatty acid does not matter, rather it is the presence of one unsaturated fatty acid that makes the triacylglycerol a monounsaturated fat. Monounsaturated fats are usually viscous liquids at room temperature, but some can be semisolids. A triacylglycerol containing two or more unsaturated fatty acids is a POLYUNSATURATED TRIACYLGLYCEROL (POLYUNSATURATED FAT). These compounds are oily liquids at room temperature. In triacylglycerols as the length of the fatty acids increase the melting point increases and as the number of carbon-carbon double bonds increase the melting point decreases.

This class of lipids serves three functions in the body:

O

O


1. They are the major energy storage depot in animals. The majority of excess energy in animals and humans is stored as triacylglycerols. An unlimited supply can be stored.

2. They serve as insulators against heat and cold. 3. They serve as shock absorbers. The internal organs are surrounded by a layer of triacylglycerol and

this fat protects the internal organs from friction and/or trauma.

Polyunsaturated fats can be converted to monounsaturated fats and saturated fats by the process of HYDROGENATION. Remember alkenes can be hydrogenated in an addition reaction where hydrogen is added across the carbon-carbon double bond forming a carbon-carbon single bond. This same reaction can be performed with polyunsaturated triacylglycerols. When hydrogenation is performed the liquid polyunsaturated fat is converted into a semisolid monounsaturated or a solid saturated fat. MARGARINE is formed by this process and so are the “bad” trans fatty acids. {Note: The structure of a triacylglycerol was given in the Lipids Handout Document. It is necessary to be able to identify the structure of this lipid when seen or when described in words.}

Phosphoglycerides

Moving up in complexity, the next group of glycerol lipids is the Phosphoglycerides. As their name implies these molecules contain a molecule of glycerol and a phosphate ion. The phosphate is in ester linkage to the hydroxyl group on carbon three of glycerol. All the molecules in this class also contain two fatty acids in ester linkage to carbon one and two of glycerol. This molecule (glycerol, two fatty acids, and phosphate) is called Phosphatidate, the simplest phosphoglyceride. The cell contains very little Phosphatidate. Phosphatidate is the precursor for the synthesis of the other phosphoglycerides. The cell converts the Phosphatidate into the other phosphoglycerides by adding one of the polar alcohols to the phosphate group. An ester linkage is formed between the hydroxyl group on the polar alcohol and the phosphate group.

From phosphatidate the cell synthesizes five major phosphoglycerides:

1. When choline is esterified (linked) to the phosphate of phosphatidate the resulting phosphoglyceride is Phosphatidylcholine. This molecule is also known a Lecithin.

2. When ethanolamine is linked to the phosphate of phosphatidate the resulting phosphoglyceride is Phosphatidylethanolamine.

3. When serine is linked to phosphatidate the resulting phosphoglyceride is Phosphatidylserine. 4. When inositol is linked to phosphatidate the resulting phosphoglyceride is Phosphatidylinositol. 5. A glycerol molecule can be esterified between two phosphatidate molecules forming

Diphosphatidylglycerol also known as Cardiolipin.

The phosphoglycerides are VERY AMPHIPATHIC molecules. They have a very polar region, a polar “head”, consisting of the phosphate ion and the polar alcohol, and they have a very non-polar, very hydrophobic region, or a non-polar “tail”composed of the hydrocarbon chains of the two fatty acids. This unique structural feature makes these molecules uniquely suitable for the task they perform. The phosphoglycerides are components of cellular membranes. Cell membranes are composed primarily of lipid and protein and the phosphoglycerides comprise a large portion of the lipids present in cellular membranes. Phosphoglycerides are not used for energy storage, rather they are part of the structures that keep the outside out, the inside in, and the various organelles separated. In addition to lipids and protein, Eukaryotic cell


membranes also contain some carbohydrate. When the structure of cell membranes is discussed the importance of the amphipathic nature of theses molecules will become apparent. Cardiolipin was first isolated from the heart. It is a major component of mitochondrial membranes and it was first isolated from heart muscle because this organ is very rich in mitochondria. {Note: The structures of the phosphoglycerides are in the Lipids Handout Document. It is necessary to be able to identify the structures of these lipids when seen or when described in words.}

Ether Lipids / Plasmalogens

Ether lipids contain a long chain alcohol bonded to the hydroxyl group on carbon three (3) of glycerol by an ether bond. Ether lipids are synthesized from the phosphoglycerides by an exchange reaction. The fatty acid on the last carbon is exchanged for a long chain alcohol. The alcohol is covalently linked to the glycerol by an ether bond. Choline or ethanolamine is the polar alcohol usually present on ether lipids. The PLASMALOGENS are a subset of the ether lipids. The plasmalogens contain an alcohol with a double bond between carbon 1 and carbon 2. The PLASMALOGENS contain a VINYL ALCOHOL. The ether lipids / plasmalogens are also components of cellular membranes. About 50% of the membrane lipids of heart muscle are either ether lipids or plasmalogens. Some of the ether lipids and plasmalogens have signal molecule / hormone like activity. For example PLATELET ACTIVATING FACTOR is an ether lipid with potent signaling activity. When released from the membranes of activated basophils, it stimulates platelet aggregation and the release of serotonin, a vasoconstrictor, from platelets. {Note: The structures of the ether lipids and plasmalogens are in the Lipids Handout Document. It is necessary to be able to identify the structures of these lipids when seen or when described in words.}

Spingolipids

The sphingolipids are the next group of saponifiable lipids. These molecules do not contain glycerol. The backbone of this group of lipids is a molecule of sphingosine.

Ceramides

The Ceramides are the simplest subgroup of sphingolipids. The ceramides contain a single fatty acid covalently linked to a molecule of sphingosine. The covalent bond joining the fatty acid to the sphingosine is an amide bond formed between the carboxyl group of the fatty acid and the amino nitrogen on carbon two of sphingosine. The cell contains very little ceramide since it is used as a precursor for the other sphingolipids. The small amount present in the cell is part of the lipids of the plasma membrane.

Sphingomyelin

The Sphingomyelins subclass of sphingolipids is synthesized from ceramide. A phosphate is covalently linked by an ester bond to the hydroxyl group on carbon one of sphingosine and then either choline or ethanolamine is covalently linked to the phosphate by a second ester bond. These groups along with the fatty acid in amide linkage to the nitrogen on carbon two are present on the molecule. The Sphingomyelins are very amphipathic molecules, they have a polar head; the phosphate, the choline or ethanolamine, the amide functional group, and the hydroxyl group on carbon 3. The non-polar region of these molecules is the hydrocarbon part of the fatty acid and the last 15 carbons of sphingosine. Sphingomyelins, as the name


implies, were first isolated from the myelin of the brain and spinal cord. These lipids are particularly rich in nervous tissue, but are found to a greater or lesser extent in the plasma membrane of all cells.

Cerebrosides

Along with the fatty acid in amide linkage on carbon two of sphingosine, the Cerebroside subclass of sphingolipids contain a single monosaccharide, either glucose or galactose in glycosidic linkage to the hydroxyl group on carbon one of sphingosine. The anomeric carbon (C1) of the sugar is made to react with the –OH group on C-1 of sphingosine. The sugar is present along with the fatty acid in amide linkage to the nitrogen on carbon two. The Cerebrosides are amphipathic molecules, they have a polar head; the glucose or galactose, the amide functional group, and the hydroxyl group on carbon 3 and a non-polar region; the fatty acid and the last 15 carbons of sphingosine. The Cerebrosides, as the name implies, were first isolated from the cerebrum of the brain. They are particularly rich in nervous tissue, but they are present to a greater or lesser extent in all cell membranes.

Gangliosides

In the Ganglioside subclass of sphingolipids a hetero-oligosaccharide containing 3 to 15 sugar residues is attached to carbon one of ceramide by glycosidic bonds. The heteropolysaccharide can contain glucose, galactose, mannose, glucosamine, galactosamine, N-acetylglucosamine, and/or N-acetylgalactosamine. The terminal (last) sugar(s) of the heteropolysaccharide is usually one or more sialic acid residues. The Gangliosides are very amphipathic molecules, they have a very polar head; the hetero-oligosaccharide, the amide functional group, and the hydroxyl group on carbon 3 and a non-polar region; the fatty acid and the last 15 carbons of sphingosine. The Gangliosides, as the name implies, were first isolated from the ganglia of the brain and spinal cord. They are particularly rich in nervous tissue, but they are present to a greater or lesser extent in all plasma membranes. For example, the three heteropolysaccharides that determine whether an individual has blood type A, blood type B, or blood type O are examples of the types of heteropolysaccharides that are attached to ceramide to form a ganglioside. Hence, the presence of one or more specific ganglioside(s) on the cell membrane of red blood cells determines whether an individual has type A, type B, type AB, or type O blood. Specific Gangliosides on other tissues need to be matched before tissue transplantation. {Note: The structures of the sphingolipids are given in the Lipids Handout Document. It is necessary to be able to identify the structures of these lipids when seen or when described in words.}

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Nonsaponifiable Lipids

The nonsaponifiable lipids cannot be hydrolyzed, broken down into smaller molecules. They do not contain ester, amide, or glycosidic bonds. The nonsaponifiable lipids are large non-polar hydrophobic molecules. There are three major classes of nonsaponifiable lipids; the fat soluble vitamins (vitamins A, D, E, and K), the eicosanoids, and the cholesterol based lipids. The cholesterol based lipids include cholesterol itself, the bile acids, salts, and esters, and the steroid hormones.

Fat Soluble Vitamins


The fat soluble vitamins are the simplest group of nonsaponifiable lipids. The fat soluble vitamins belong to a class of molecules called ISOPRENES or TERPENES. These molecules are synthesized from an unsaturated five carbon precursor, ISOPRENE (2-methyl-1,3-butadiene). There are four fat soluble vitamins; Vitamin A, Vitamin D, Vitamin E, and Vitamin K.

Vitamin A

There are three active forms of Vitamin A; Retinol, Retinal, and Retinoic Acid. Retinal is necessary for vision. In the retina of the eye it is covalently linked to a light sensitive protein, opsin or rhodopsin. Retinol plays a role in the regulation of gene expression during cell differentiation especially during embryonic and fetal development. Retinoic Acid helps to maintain the integrity of the skin. Vitamin A can also used by cells as an antioxidant. It reacts with strong oxidizing agents produced in the cell from molecular oxygen and prevents these very reactive oxidizing agents from destroying cell membranes and other necessary cellular components. The antioxidant role is a minor function. Vitamin A can be obtained from animal sources directly or from the hydrolysis of the 40 carbon molecule, β-carotene, obtained from yellow/orange vegetables. The lack of Vitamin A in the diet first results in Night Blindness, followed by other Vision Abnormalities and a “Failure to Thrive”.

Vitamin D

Vitamin D is a group of related lipids. Vitamin D3, also known as cholecalciferol, is formed nonenzymatically by sunlight reacting with the cholesterol derivative, 7-dehydrocholesterol, present in the skin. The active form of vitamin D, 1,25-Dihydroxycholecalciferol, is formed from vitamin D3 by two hydroxylation reactions, by the addition of two hydroxyl groups to the molecule. The active vitamin plays a role in maintaining normal plasma Ca+2 levels. It stimulates the uptake of Ca+2 from the intestine, the reabsorption of Ca+2 by the kidney, and the uptake of Ca+2 from bone. (Note: Vitamin D is one of many biomolecules that can be classified in more than one way. It is a fat soluble vitamin and it is a derivative of cholesterol. Hence, it could be placed with cholesterol and the lipids derived from cholesterol.)

Vitamin E

Vitamin E, or α-Tocopherol is believed to function as a scavenger of strong oxidizing agents, as an antioxidant. Its antioxidant action prevents damage to unsaturated fatty acids in biological membranes.

Vitamin K

Vitamin K is a lipid vitamin obtained from plants. It is required for the normal synthesis of some of the proteins involved in the blood coagulation system. It is the only fat soluble vitamin that has cofactor activity. A cofactor is a molecule necessary for the normal function of an enzyme. A deficiency in Vitamin K results in abnormal blood clotting. The affected individual bleeds an abnormally long time and in extreme cases could exsanguinate (bleed to death). The anticoagulant drug Coumadin is given to individuals that have had thrombotic events. A thrombotic event occurs when the blood clots abnormally,


Isoprene

causing pathological states such as a heart attack or a stroke. Coumadin antagonizes the action of Vitamin K, it inhibits the vitamin from acting as a cofactor during the synthesis of the blood coagulation factors. Individuals on this drug are tested regularly to insure that they do not bleed excessively.

Eicosanoids

EICOSA means 20. All the Eicosanoids are derived from the 20 carbon fatty acid, Arachidonate. Arachidonate is the precursor for the Eicosanoids. The Eicosanoids are local hormones, autocrine or paracrine. They are synthesized by a certain cell type, released from that cell, and they evoke a specific response in neighboring cells or in a very localized region of the body. Their effect is much more short range than endocrine hormones which produce an effect throughout the body. The Eicosanoids are divided into three groups depending upon common structural characteristics. The three groups of eicosanoids are the Prostaglandins, Thromboxanes, and Leukotrienes. The eicosanoids produce a variety of biological effects. They:

1. cause smooth muscle contractions. 2. cause smooth muscle relaxation. 3. inhibit the secretion of HCl by the stomach. 4. inhibit the use of fatty acids for energy. 5. inhibit blood clotting. 6. stimulate blood clotting. 7. regulate the synthesis of steroids 8. regulate nervous transmission. 9. mediate (increase or decrease) the sensation of pain. 10. mediate (increase or decrease) the inflammatory response.

For example endothelial cells, the cells that line the blood vessels, continually produce a group of Prostaglandins that prevent blood coagulation. When a “boo-boo” happens these cells are damaged or destroyed and the production of these compounds in a localized region drops to zero. The lack of these compounds at the site of a wound allows platelets to bind to the site, forming a platelet plug that initially stops blood loss. Platelets produce Thromboxanes that simulate the blood coagulation system. A blood clot (scab) forms sealing the wound until healing occurs.

Cholesterol

Cholesterol and the biomolecules synthesized from Cholesterol are the last group of non saponifiable lipids. Like the fat soluble vitamins Cholesterol is an isoprene. Cholesterol is an important component of cell membranes, without it cell membranes would not function properly. Cholesterol is also converted into a variety of important biomolecules. It is converted into the Bile Acids, Bile Salts, Bile Esters, and it is converted into the Steroid Hormones. The Bile Acids, Bile Salts, and Bile Esters are important and necessary for the normal digestion and absorption of lipids.

There are three classes of Steroid Hormones that utilize Cholesterol as precursor. The first group of steroid hormones synthesized from cholesterol are the Progestins. The Progestins (e.g. progesterone) readies the female uterus for pregnancy, stimulates ovulation, and maintains pregnancy during the early stages. The


Progestins are the precursors for the other classes of steroid hormones. They are converted into the Glucocorticoids (e.g. cortisol). The Glucocorticoids are one of several hormones that helps maintain blood glucose level and they modulate the inflammatory response to injury. The progestins are also converted into the Mineralocorticoids (e.g. aldosterone). The Mineralocorticoids helps to control the monovalent ion (Na+, K+, HCO3–, and Cl–) concentrations in the body. Glucocorticoids and mineralocorticoids are synthesized and secreted by the cortex of the Adrenal Gland. Finally, the progestins can be converted into the Androgens (e.g. testosterone). These hormones stimulate/mediate sexual maturation in both sexes, but especially the male. The androgens are the precursors to the Estrogens (e.g., β-estradiol). These hormones stimulate/mediate sexual maturation in both sexes, but especially the female. The androgens and estrogens are classified as anabolic steroids. {Note: The structures of the nonsaponifiable lipids are given in the Lipids Handout Document. It is NOT necessary to be able to identify the structures of these lipids. Know their functions}

The French Paradox (FYI) A diet high in saturated fats (saturated triacylglycerols) has been implicated as a cause for cardiovascular disease. It has been suggested that Americans limit the amount of fat in their diets. The French diet is as rich or richer in saturated fats when compared to the American diet and yet the French have a lower incidence of cardiovascular disease – The French Paradox. Biochemists, nutritionists, and physicians looked closely at the French diet and noted that one of the major differences between their diet and the American diet is the amount of red wine consumed by the French. When the red wine was examined it was found to contain relatively high concentrations of a groups of compounds called Polyphenols, the most abundant of these polyphenols is the molecule Resveratrol. Resveratrol can be found in high concentrations in the skins of red wine grapes, peanuts, mulberries, chocolate and several other fruits and nuts. The plant produces this compound as a fungicide, it destroys fungi that invade the fruit and cause it to rot. In animals Resveratrol, and the other polyphenols, have been shown to be antioxidants, anticoagulants, anti-inflammatory agents, to have anticancer effects and to increase high-density lipoprotein (HDL), the “good cholesterol”. It has been difficult to isolate and purify these compounds because they are easily oxidized and destroyed by the oxygen in the air. Resveratrol has become available on the internet and in “health food stores” as a dietary supplement. However, its potency and effectiveness is open to question.

Membranes - THE FLUID MOSAIC MODEL OF MEMBRANES

Membranes surround and delineate the cell. In eukaryotic cells membranes also surround and delineate the subcellular organelles. They function to keep the inside in and the outside out. Membranes are supramolecular complexes of phosphoglycerides, sphingolipids, cholesterol and proteins. How the lipid components of the membrane are assembled into the complex structure will be examined now. Proteins will be discussed shortly. Once the structure of proteins has been discussed the structure and organization of membranes will be revisited to look at how membrane proteins control the flow materials and information into and out of the cell.


O H

O H

H O

Resveratrol

Four biological macromolecules are assembled into the supramolecular complex of the cell membrane. The four macromolecules are the Phosphoglycerides, Sphingolipids, Cholesterol and Proteins.

How are the lipids assembled into a membrane?

The phosphoglyceride and sphingolipid part will be examined first. What would happen if a mixture of phosphoglycerides and sphingolipids were placed in/on water or in/on a buffer? The molecules in this mixture would align themselves on the surface of the water or buffer such that the polar head of these molecules would interact with the polar water or buffer as well as with each other, and the non-polar ends, the non-polar tails would interact with each other and the nonpolar air. Non-polar regions of the molecule (the non-polar tails) would arrange themselves to get as far away from the polar water as possible.

The lipid and water mixture is now rapidly agitated and then allowed to settle. What has agitation done to this mixture of lipids?

Some of the lipids will remain at the water / air interface with their polar heads interacting with water and their tails interacting with air.

Some of the lipids will form micelles similar in structure to the micelles formed by soaps.

Some of the lipids will form a two layered structures enclosing some of the water/buffer. The polar heads of the outer layer of lipids interacts with the exterior water and the polar heads of the inner layer of lipids interacts with the water/buffer trapped inside the structure. The non-polar tails of the two lipid layers interact with each other in the middle of this two layered structure. This two layered structure is called a LIPID BILAYER and it is a major part of a cell membrane.

Lipid Bilayer

Lets look at the structure of the lipid bilayer as it is found in cellular membranes. All membranes contain phosphoglycerides, sphingolipids, and cholesterol. Both layers, both leaflets of the membrane contain phosphoglycerides and cholesterol. Only the outer layer of the lipid bilayer, the outer leaflet, the outside surface of the cell contains sphingolipids.

Sidedness - Telling the Outside from the Inside

The cellular membrane, the plasma membrane, is a lipid bilayer. The outside layer of the lipid bilayer, the layer in contact with extracellular water is termed the outer leaflet. The layer in contact with intracellular water, the inner layer of the bilayer is called the inner leaflet. Plasma membranes have a definite sidedness, a definite orientation. The the outer leaflet can be distinguished from the inner leaflet based upon phospholipid composition. All of the Cerebrosides and Gangliosides and the majority of the Sphingomyelins are found in the outer leaflet of the membrane. The monosaccharide molecule attached to the cerebrosides and the hetero-oligosaccharides attached to the gangliosides orient these molecules correctly in the membrane. The polysaccharide is directed extracellularly, directed toward the outside of the


cell. (Remember, one of the functions of heteropolysaccharides is to orient molecules correctly in cellular membranes.) For most mammalian cells, the outer leaflet has a higher proportion (> than 50% of the total) of Phosphatidylcholine, whereas the inner leaflet is rich (> than 50% of the total) in Phosphatidylserine, Phosphatidylinositol, and Phosphatidylethanolamine.

Forces

Weak intermolecular interactions hold the lipids of the membrane together. Three forces stabilize the membrane structure. The first force is hydrogen bonding and/or dipole-dipole interactions between the polar ends of the lipids and the polar water molecules inside and outside of the cell. The second force is hydrogen bonding and/or dipole-dipole interacts between the polar heads of the amphipathic lipids within an individual layer; i.e., within the inner layer or within the outer layer, not across the layers. The third force is the London forces between the non-polar hydrophobic tails of these amphipathic lipid molecules. These London forces are HYDROPHOBIC INTERACTIONS because the molecules interacting with each other are all non-polar hydrophobic molecules. The hydrophobic interactions between the non-polar “tails” contribute the largest force toward stabilizing and maintaining the lipid bilayer structure.

Fluidity

Cellular membranes are fluid in nature. Lipids in the individual layers migrate within that layer and the outer layer migrates, flows (moves) over the inner leaflet. For example a lipid molecule in the outer leaflet migrates rapidly throughout the outer leaflet. It can move from one end of the cell to the other in a few seconds. Lipid molecules rarely move between layers, a lipid molecule in the outer layer rarely “flip-flops” into the inner layer and/or visa versa.

Cellular membranes must be fluid to perform their function. Since life forms are found over a wide range of temperatures, membranes must be fluid over this wide range of temperatures. The fluidity of cellular membranes is controlled by two factors. First, it is controlled by the ratio of saturated to unsaturated fatty acids in the membrane lipids. Increasing the amount of saturated fatty acids decreases the fluidity, whereas increasing the concentration of unsaturated fatty acids increases the fluidity. The “cis” double bonds in the unsaturated fatty acids puts a “kink” in the molecule and the “kinky” unsaturated fatty acids do not allow the lipid molecules to pack together well. Bacteria and animals that live in cold climates have membrane lipids (phosphoglycerides and sphingolipids) with a high proportion of unsaturated fatty acids; the “kinky” fatty acids have low melting points and therefore the membranes remain fluid. The bacteria and animals that live in hot climates have membrane lipids with a high proportion of saturated fatty acids; the “straight” fatty acids have high melting points. Second, it is controlled by the amount of cholesterol “dissolved” in the membrane. Increasing the amount of cholesterol in the membrane increases membrane fluidity by broadening the temperature at which a membrane undergoes the transition from fluid state to solid state.

Barrier Properties

The lipid layer of the membrane is a very effective barrier against molecules entering or leaving a cell. The polar surfaces on the exterior and interior of the membrane prevents the movement of non-polar molecules into or out of the cell. Non-polar molecules do not interact (well) with the polar heads of the membrane lipids. The non-polar middle region of the membrane prevents the movement of polar molecules. Polar


molecules do not interact (well) with the non-polar tails of the membrane lipids. A few small non-polar molecules can freely diffuse across a cell membrane. N2, O2, CO2, and urea, can freely pass across the cell membrane. A small amount of water can pass into and out of the cell through the membrane. The movement of water across the cell membrane is called OSMOSIS; the movement of solute molecules such as N2 O2, CO2, and urea across the membrane is called DIFFUSION.

Question - If the membrane is such an effective barrier against molecular movement into or out of the cell, how do important biomolecules enter and/or leave the cell? How does information enter and/or leave the cell?

The cell has to get large polar and non-polar molecules in and out. It needs glucose, lipids, and amino acids for energy and growth, yet these molecules are too large or too polar to cross the cell membrane and enter the cell. These molecules enter the cell using the membrane proteins. {The mechanism of to how molecules and information get into and out of the cell will be discussed after proteins are examined.}

There are three types of proteins associated with or present in the cell membrane. EXTRINSIC or PERIPHERAL membrane proteins are bound to and interact with only one surface of the membrane. These proteins bind to and interact with the polar heads of the membrane lipids by hydrogen bonding and/or dipole-dipole interactions. Cytoskeleton proteins, antigenic determinants, and cell-cell contact molecules are examples of extrinsic (peripheral) proteins.

The second type of membrane proteins are the INTRINSIC, INTEGRAL, or TRANSMEMBRANE proteins. These proteins are embedded within the membrane and extend completely through it. Parts of the protein are exposed on both the inside and outside of the cell. The parts of the protein that interact with the polar surfaces of the membrane interact by dipole-dipole interactions and/or hydrogen bonding. The parts of the protein that interact with the non-polar regions of the membrane do so by hydrophobic interactions. Molecular transporters and hormone receptors are examples of intrinsic (integral) proteins. They can also be antigenic determinants, cell-cell contact molecules, and anchors for the cytoskeleton.

The third type of proteins associated with the cell membrane are LIPID LINKED proteins. These proteins are covalently linked to a fatty acid, a phosphoglyceride, or a complex isoprene. The lipid moiety is inserted into either the inner leaflet or the outer leaflet of the membrane and the protein is tethered to the surface of the cell by this lipid molecule. Lipid linked protein have the most freedom of movement of all the membrane associated proteins.

The extrinsic membrane proteins associated with the outer leaflet (the outside of the cell) and many intrinsic membrane proteins are usually GLYCOPROTEINS. GLYCOPROTEINS are protein molecules with one or more hetero-oligosaccharides or heteropolysaccharides covalently attached. The sugar part of the molecule signals to the cell that these proteins are to be embedded in the cell membrane and they orient the protein correctly in the cell membrane. The heteropolysaccharide is always on the exterior of the cell (pointing out) where it can interact, form hydrogen bonds, with extracellular water.

Since the proteins are embedded in the membrane and interact with the membrane lipids these embedded proteins move along the cell surface as the membrane lipids move. The membrane is a mosaic of lipids and proteins in constant lateral motion.


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