Lipid Biochemistry (Part II)

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Simple lipids: Fats and oils 

(Storage lipids)

These are triglycerides or triacylglycerols.

These are esters of 3 fatty acid molecules with trihydroxy alcohol, glycerol.

A fat is solid at room temperature while oil is liquid.

Triglycerides are most abundant among all lipids, constituting about 98% of total dietary lipids.

They are major storage components or depot fats in plant and animal cells but are not normally found in membranes.

They are non-polar, hydrophobic molecules since they contain no electrically charged or highly polar functional groups.

Triglycerides which contain the same kind of fatty acids in all three positions are called simple triacylglycerols and are named after the fatty acid they contain. 

Simple triacylglycerols of 16:0, 18:0, and 18:1, for example, are tristearin, tripalmitin, and triolein, respectively.

Triglycerides which contain two or more different fatty acids are called mixed triacylglycerols.

To name these compounds unambiguously, the name and position of each fatty acid must be specified.

Functions of fats and oils

In eukaryotic cells, triacylglycerols form a separate phase of microscopic, oily droplets in the aqueous cytosol, serving as depots of metabolic fuel.

Adipocytes or fat cells store large amounts of triacylglycerols as fat droplets.

Triacylglycerols are also stored as oils in the seeds of many types of plants, providing energy and biosynthetic precursors during seed germination.

Adipocytes and germinating seeds contain lipases that catalyze the hydrolysis of stored triacylglycerols, releasing fatty acids for export to sites where they are required as fuel.

Humans have adipocytes under the skin, in the abdominal cavity, etc.

Seals, walruses, penguins, and other warm-blooded polar animals are amply padded with triacylglycerols which provide insulation.

In hibernating animals, the huge fat reserves accumulated before hibernation serve the dual purposes of insulation and energy storage.

In sperm whales, a store of triacylglycerols and waxes (low density) allows the animals to match the buoyancy of their bodies to that of their surroundings during deep dives in cold water.

Simple lipids: waxes 

(Energy Stores and Water Repellents)

Waxes are esters of fatty acids with high molecular weight monohydroxy alcohols.

Fatty acids range between C14 and C36 while alcohols range from C16 to C36.

Melting varies from 60° - 100ºC.

The term ‘wax’ originated from old English ‘weax’ meaning ‘material of the honeycomb’.

Carnauba wax is the hardest known wax. It consists of fatty acids esterified with tetracosanol [CH₃(CH₂)₂₂CH₂OH] and tetratriacontanol [CH₃(CH₂)₃₂CH₂OH].

Waxes are unusually inert due to their saturated nature of the hydrocarbon chain. However, they can be slowly split with hot alcoholic KOH.

Functions of waxes

In planktons, the chief storage form of metabolic fuel.

Certain skin glands of vertebrates secrete waxes to protect hair and skin and keep it pliable, lubricated, and waterproof.

Some aquatic birds, secrete waxes from their preen glands to keep their feathers water-repellent.

Leaves of many tropical plants are coated with a thick layer of waxes, which prevents excessive evaporation of water and protects against parasites.

Biological waxes find a variety of applications in the pharmaceutical, cosmetic, and other industries. 

Lanolin (from lamb’s wool), beeswax, carnauba wax (from a Brazilian palm tree), and wax extracted from spermaceti oil (from whales) are widely used in the manufacture of lotions, ointments, and polishes.

Compound lipids: Phospholipids 

(Structural lipids)

Phospholipids are the most abundant membrane lipids.

They serve primarily as structural components of membranes and are never stored in large quantities.

Phospholipids contain phosphorus in the form of phosphoric acid groups. They differ from triglycerides in possessing usually one hydrophilic polar ‘head’ group and usually two hydrophobic nonpolar ‘tail’ groups. They are often called polar lipids. Thus, phospholipids are amphipathic.

In phospholipids, two of the –OH groups in glycerol are linked to fatty acids while the third –OH group is linked to phosphoric acid. The phosphate is further linked to one of a variety of small polar head groups (alcohols).

Phospholipids can be classified into:

Phosphoglycerides

Phosphoinositides

Phosphosphingosides

Phosphoglycerides

Major phospholipids found in membranes.

It consists of two fatty acid molecules or ‘tails’ esterified with the first and second –OH groups of glycerol.

The third –OH group of glycerol forms an ester bond with phosphoric acid. An additional substituent group is esterified with the phosphoric acid. This is referred to as ‘head group’ as it is present at one end of the long phosphoglyceride molecule.

Of the two fatty acid molecules, the one on C1 is saturated (C16-C18) while the one on C2 is unsaturated (C18-C20).

C2 of glycerol is asymmetric in nature.

All phosphoglycerides contain a negative charge on the phosphoric acid at pH 7. In addition, the head group may also have one or more electrical charges at pH 7.

Phosphoglycerides are of 3 types:

Lecithins (Phosphatidyl cholines)

Cephalins (Phosphatidyl ethanolamine and phosphatidyl serine)

Plasmalogens (Phosphoglyceracetals)

Lecithins (Phosphatidyl cholines)

Found in various oil seeds like soybeans and yeasts. In animals, glandular and nervous tissues are rich in lecithins.

Required for normal transport and utilization of other lipids in the liver. In its absence, accumulation of lipids occurs in the liver to as much as 30% (against 3-4% in normal) giving rise to a condition called ‘fatty liver’. This may lead to fibrotic changes.

Lecithins contain two fatty acids esterified with any two –OH groups of glycerol while the third –OH group is esterified with a phosphoric acid group. The phosphoric acid group is again linked to a nitrogen base, choline.

Hydrolysis of lecithins

On complete hydrolysis, lecithins yield a mixture of choline, phosphoric acid, glycerol, and two moles of fatty acids.

But partial hydrolysis of lecithins by lecithinase (active components of snake venom) causes removal of one fatty acid to yield lysolecithins.

When subjected to the bloodstream (as a result of snakebite), lysolecithins cause rapid rupture of RBC (hemolysis).

Cephalins

These are closely associated with lecithins in animal tissues. They are also structurally similar to lecithins except that the choline is replaced by either ethanolamine or serine.

Accordingly, two types of cephalins are recognized:

Phosphatidyl ethanolamine

Phosphatidyl serine

Since the primary amino group is a weaker base than the quaternary ammonium group of choline, the cephalins are more acidic than lecithins. Cephalins are also comparatively less soluble in alcohol than lecithins.

Snake venoms containing lecithinase can also split off fatty acids from cephalins leaving hemolytic lysocephalins.  

Plasmalogens (phosphoglyceracetals)

Plasmalogens constitute about 10% of phospholipids in the brain and muscles. About half of heart phospholipids are plasmalogens.

These ether phospholipids are common in membranes of halophilic bacteria because they are resistant to phospholipases.

Structurally they are similar to lecithins and cephalins but have one of the fatty acid chains replaced by an unsaturated ether.

Since nitrogen base can be choline, ethanolamine, or serine, plasmalogens can be of three types:

Phosphatidal choline

Phosphatidal ethanolamine

Phosphatidal serine

Phosphoinositides (phosphatidyl inositols)

Phosphoinositides have been found to occur in phospholipids of brain tissues and soybeans. They play an important role in the transport process as well as a signaling intermediate in cells.

They have a cyclic hexahydroxy alcohol called inositol which replaces the base. The inositol is present as the stereoisomer, myo-inositol.

Number of phosphate groups may be one, two, or three. Accordingly, mono-, di- and triphosphoinositides are found.

Phosphosphingosides (sphingomyelins)

Commonly found in nerve tissues, especially in the myelin sheaths. Absent in plants and microorganisms.

In a syndrome called Niemann-Pick disease, the sphingomyelins are stored in the brain in large quantities.

They lack glycerol and instead contain sphingosine or a closely related dihydrosphingosine.

They are electrically charged molecules and contain phosphocholine as a polar head group.

Functions of phospholipids

In association with proteins, phospholipids form the structural components of membranes and regulate membrane permeability.

Phospholipids in the mitochondria maintain the conformation of electron transport chain components and thus cellular respiration.

They participate in the absorption of fats from the intestine.

They are essential for the synthesis of different lipoproteins and thus participate in the transport of lipids.

They prevent the accumulation of fats in the liver (lipotropic factors).

They participate in the transport of cholesterol and thus help in the removal of cholesterol from the body.

They act as surfactants (respiratory distress syndrome).

Cephalin participates in blood clotting.

Phosphatidyl inositol is the source of the second messenger that is involved in the action of some hormones.

Lipid Biochemistry (Part I)

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Lipids

Lipids are water-insoluble biomolecules that are highly soluble in organic solvents like chloroform.

Lipids are a heterogeneous group of compounds related to fatty acids and include fats, oils, waxes, and other related substances.

Lipids are hydrophobic in nature.

The term ‘lipid’ was first coined by a German biochemist, Bioor, in 1943.

Chemically fats are defined as the esters of glycerol and fatty acids or triglycerides of fatty acids.

Functions of lipids

A diverse range of functions in biological systems:

Important constituents of diet because of their high energy value

Serve as a source of fat-soluble vitamins and essential fatty acids in natural foodstuffs.

Serve as a stored form of energy in adipose tissues.

Serve as an insulating material in subcutaneous tissues and around certain organs.

Lipoproteins are important constituents of biological membranes.

Plays crucial roles as:

enzyme cofactors, 

electron carriers, 

light absorbing pigments, 

emulsifying agent in digestive tracts, 

hormones and intracellular messengers

May act as chaperones to help membrane proteins fold.

Advantage of energy storage as fatty acids

The carbons in the fatty acids (mostly –CH₂ group) is almost completely reduced compared to the carbon in other simple biomolecules (sugars, amino acids, etc.). Therefore, oxidation of fatty acids will yield more energy (in form of ATP) than any other form of carbon.

Fatty acids are not hydrated as mono- and polysaccharides and thus can be packed more closely in storage tissues.

Fatty acids

Fatty acids are hydrocarbon chains of various lengths and degrees of unsaturation that terminate with the carboxyl group.

Fatty acids that occur in natural fats are usually monocarboxylic.

They contain an even number of carbon atoms as they are synthesized from condensation of 2 carbon units. 

These range from 4 to 36 C-atoms.

The chain may be saturated (no double bonds) or unsaturated (contain one or more double bonds).

Some fatty acids may have hydroxyl groups in the chain (hydroxy-fatty acid) and still, others may possess ring structures (cyclic-fatty acids).

Alcohols in lipid molecules

Alcohols present in lipid molecules commonly include glycerol, cholesterol, and higher alcohols like cetyl alcohol and mericyl alcohol.

In the structural formula of glycerol, the C-atoms are numbered 1, 2, 3 from any end. Since C1 and C3 are identical, they are also denoted as α, β, and α’.

Nomenclature of fatty acids

The systematic nomenclature of fatty acids is based on the Genevan System under the instruction of IUPAC.

The fatty acid is named after the hydrocarbon with the same number of carbon atoms, the suffix –oic is written in place of the final letter ‘-e’ in the name of the hydrocarbon.

The names of the saturated fatty acids end with the suffix –anoic and those of unsaturated fatty acids with the suffix –enoic.

The positions of the carbon atoms are denoted by numbering (carboxyl carbon atom is C1, the adjacent carbon atom is C2 and so on) or by using Greek letters (C2 is denoted as α-carbon, C3 is denoted as β-carbon and so on).

A widely used convention is to indicate the carbon atom number followed by the number and position of double bonds in the case of unsaturated fatty acids (position of a double bond is indicated by a lower number of the two carbon atoms involved in double bonding).

In nearly all naturally occurring unsaturated fatty acids, the double bonds are in the cis configuration.

Trans fatty acids are produced by fermentation in the rumen of dairy animals and are obtained from dairy products and meat.

They are also produced during hydrogenation of fish or vegetable oils. 

Diets high in trans fatty acids correlate with increased blood levels of LDL (bad cholesterol) and decreased HDL (good cholesterol).

French fries, doughnuts, fast foods, and cookies tend to be high in trans fatty acids.

Lipid classification

Bioor (1943) classified lipids on the basis of their chemical composition:

Simple lipids or homolipids

Compound lipids or heterolipids

Derived lipids

Conn and Stumpf (1976) traditionally classified lipids into the following categories:

Acyl glycerols

Waxes

Phospholipids

Sphingolipids

Glycolipids

Terpenoid lipids including carotenoids and steroids

Lipid types (on the basis of function)

Physical properties of lipids

State: Saturated fatty acids are solid at room temperature while unsaturated fatty acids are liquid, in general.

Colour, odour and taste: Pure fats are colourless, odourless and bland taste.

Solubility: Soluble in organic solvent. Longer the fatty acyl chain and fewer the double bonds, the lower is its solubility in water.

Specific gravity: Less than 1

Geometric isomerism: Present

Emulsification: Found

Melting point: Depends on the chain length of the constituent fatty acyl chain and the degree of unsaturation.

Chemical properties of lipids

Hydrolysis: Fats are hydrolyzed by enzyme lipase to yield fatty acids and glycerol.

Saponification: Hydrolysis of fats by alkali is called saponification which results in the formation of glycerol and salts of fatty acids (called soaps).

Hydrogenation: Unsaturated fatty acids react with gaseous hydrogen to yield saturated fatty acids.

Halogenation: Unsaturated fatty acids and their esters can take up halogens (Br2, I2, etc.) at their double bonds at room temperature in acetic acid or methanol solution. This reaction is the basis of ‘iodine number determination’.

Oxidation: Unsaturated fatty acids are susceptible to oxidation at their double bonds.

Acid number: It is the number of milligrams of KOH required to neutralize the free fatty acids present in 1 gram of fat. The acid number indicates the quantity of free fatty acid present in fat.

Saponification number: It is the number of milligrams of KOH required to saponify 1 gram of fat. The saponification number provides information on the average chain length of the fatty acids in fats.

Iodine number: It is the number of grams of iodine absorbed by 100 grams of fat. The iodine number indicates the degree of unsaturation of the fatty acids present in fat.

Acetyl number: It is the number of milligrams of KOH required to neutralize the acetic acid obtained by saponification of 1 gram of fat after it has been acetylated. The acetyl number indicates the number of –OH groups present in fat.

Rancidity: When lipid-rich foods are exposed for too long in the air (oxygen), they may spoil and become rancid. The unpleasant taste and smell associated with rancidity results from oxidative cleavage of the double bonds in unsaturated fatty acids, producing aldehydes and carboxylic acids of shorter chain length which are volatile.

Essential fatty acids

Our body can synthesize most of the fatty acids except a few. These fatty acids must be supplied through diet and are termed essential fatty acids.

Example-α-Linolenic acid (ω-3 fatty acid), Linoleic acid (ω-6 fatty acid)

It is recommended that essential fatty acids make up 3% to 6% of your daily caloric intake. Of this percentage, you should consume 2 to 4 times more omega-6 fatty acids than omega-3 fatty acids. 

Omega-3 sources include:

Nuts

Soybeans

Walnut oil

Canola oil

Flaxseed oil

Cold water fatty fish such as salmon, herring, cod, flounder, tuna, bluefish, and shrimp

Omega-6 sources include:

Leafy vegetables

Seeds

Nuts

Grains

Vegetable oils (corn, safflower, soybean, cottonseed, sesame, sunflower)

Roles of Essential fatty acids

• Help with cellular development and the formation of healthy cell membranes.
• Blocks tumor formation and growth of cancer cells
• Assist in the development and function of the brain and nervous system.
• Regulates proper thyroid and adrenal activity. 
• Plays a role in thinning your blood, which prevents blood clots, heart attacks and stroke.
• Regulates blood pressure, immune responses and liver function.
• Deficiency causes skin problems, including eczema, dandruff, split nails and brittle hair.
• Forms Lipid rafts which affects cellular signalling.
• Acts on DNA, activates or inhibits transcription factors.

LINKAGE, CROSSING-OVER AND CHROMOSOME MAPPING (Part I)

 

LINKAGE AND CROSSING OVER

žGenes that are on the same chromosome travel through meiosis together. This is called linkage.

žAlleles of chromosomally linked genes can be recombined by crossing over.

žDuring meiosis, homologous chromosomes pair, and physical exchange of chromosome segments recombine genes. This is supported by the cytological observation that homologous chromosomes pair during meiosis.

žAt the switch points, the two homologues are crossed over, as if each has been broken and then reattached to its partner.

žA crossover point is called a chiasma (plural, chiasmata). Crossing over describes the process that creates the chiasmata—that is, the actual process of exchange between paired chromosomes.

žRecombination—the separation of linked genes and the formation of new gene combinations—is a result of the physical event of crossing over.

Linkage

žWhen two or more characters of parents are transmitted together to the offsprings of few generations such as F1 , F2 , F3 etc. without any recombination, they are called linked characters and the phenomenon is called linkage.

žThis is a deviation from the Mendelian principle of independent assortment.

žMendel’s law of independent assortment is applicable to the genes that are situated in separate chromosomes. When genes for different characters are located in the same chromosome, they are tied to one another and are said to be linked.

žThey are inherited together by the offspring and will not be assorted independently.

žThus, the tendency of two or more genes of the same chromosome to remain together in the process of inheritance is called linkage.

Coupling vs. Repulsion

žThe condition of having the dominant alleles for both genes on the same parental chromosome and both recessive alleles on the other parental chromosome is called “coupling”.

žThe opposite condition, having one dominant and one recessive on each parental chromosome is called “repulsion”.

EARLY EVIDENCE FOR LINKAGE AND RECOMBINATION: W. Bateson and R. C. Punnett Experiment

žPlants with red flowers and long pollen grains were crossed to plants with white flowers and short pollen grains.

žAll the F1 plants had red flowers and long pollen grains, indicating that the alleles for these two phenotypes were dominant.

žWhen the F1 plants were self-fertilized, instead of the 9:3:3:1 ratio expected for two independently assorting genes, they obtained a peculiar ratio of 24.3:1.1:1:7.1.

žAmong the F2 plants, the classes that resembled the original parents (called the parental classes) are significantly overrepresented and the two other (non-parental) classes are significantly underrepresented.

žBateson and Punnet could not provide the correct explanation for this observation.

žThe correct explanation for the lack of independent assortment in the data is that the genes for flower color and pollen length are located on the same chromosome—that is, they are linked.

EARLY EVIDENCE FOR LINKAGE AND RECOMBINATION: 

MORGAN’s Experiment

žLater, Thomas Hunt Morgan found a similar deviation from Mendel’s second law while studying two autosomal genes in Drosophila.

žOne of the genes affected eye color:

pr: purple (recessive)         

pr+: red (dominant)

žThe other gene affected wing length:

vg: vestigial (recessive)         

Vg+: normal (dominant)

žMorgan performed a cross to obtain dihybrids, then followed with a testcross:

žMorgan’s testcross results were as follows (listed as the gametic classes from the dihybrid):

žThese numbers deviate drastically from the Mendelian prediction of a 1:1:1:1 ratio. Parental classes are overrepresented while the non-parental classes are underrepresented.

žThe ratio of two parental classes is 1:1 while the ratio of two non-parental classes is also 1:1.

žMorgan performed another cross to obtain dihybrids, then followed with a testcross, where the alleles were in different combinations:

žMorgan’s testcross results were as follows (listed as the gametic classes from the dihybrid):  

žAgain, these numbers deviate drastically from the Mendelian prediction of a 1:1:1:1 ratio. Parental classes are overrepresented while the non-parental classes are underrepresented.

žThe ratio of two parental classes is 1:1 while the ratio of two non-parental classes is also 1:1.

žMorgan suggested that the two genes in his analyses are located on the same pair of homologous chromosomes, i.e. they are linked.

New combinations of alleles arise from crossovers

žThe linkage hypothesis explains why allele combinations from the parental generations remain together—because they are physically attached by the segment of chromosome between them.

žBut how do we explain the appearance of the minority class of non-parental combinations?

žWhen homologous chromosomes pair in meiosis, the chromosomes occasionally break and exchange parts in a process called crossing over. The two new combinations are called crossover products.

Cis- and trans conformation

žWhen the two dominant or wild-type alleles are present on the same homolog, the arrangement is called a cis conformation.

                                              Cis:   RL/rl

žWhen the two dominant or wild-type alleles are present on the different homologs, the arrangement is called a trans conformation.

                                           Trans:   Rl/rL

CROSSING OVER AS THE PHYSICAL BASIS OF RECOMBINATION

žRecombinant gametes are produced as a result of crossing over between homologous chromosomes.

žThis process involves a physical exchange between the chromosomes.

žThe exchange event occurs during the prophase of the first meiotic division.

žAlthough four homologous chromatids are present, forming a tetrad, only two chromatids cross over at any one point.

žEach of these chromatids breaks at the site of the crossover, and the resulting pieces reattach to produce the recombinants.

žThe other two chromatids are not recombinant at this site.

žThere is a possibility for multiple exchanges in a tetrad of chromatids.

žThere may, for example, be two, three, or even four separate exchanges—customarily called double, triple, or quadruple crossovers.

žHowever, that exchange between sister chromatids does not produce genetic recombinants because the sister chromatids are identical.

EVIDENCE THAT CROSSING OVER

CAUSES RECOMBINATION

žIn 1931 Harriet Creighton and Barbara McClintock obtained evidence that genetic recombination was associated with a material exchange between chromosomes.

žCreighton and McClintock studied homologous chromosomes in maize that were morphologically distinguishable.

žTwo forms of chromosome 9 were available for analysis: one was normal and the other had cytological aberrations at each end—a heterochromatic knob at one end and a piece of a different chromosome at the other.

žThese two forms of chromosome 9 were also genetically marked to detect recombination.

žOne marker gene controlled kernel color (C, colored; c, colorless), and the other controlled kernel texture (Wx, starchy; wx, waxy).

žCreighton and McClintock performed the following testcross:

žTheir results showed that the C Wx and c wx recombinants carried a chromosome with only one of the abnormal cytological markers.

žThe other abnormal marker had evidently been lost through an exchange with the normal chromosome 9 in the previous generation. 

žThese findings suggest that recombination was caused by a physical exchange between paired chromosomes.

CHIASMATA AND CROSSING OVER

žThe cytological evidence for crossing over can be seen during late prophase of the first meiotic division when the chiasmata become clearly visible.

žAt this time paired chromosomes repel each other slightly, maintaining close contact only at the centromere and at each chiasma.

žAs we might expect, large chromosomes typically have more chiasmata than small chromosomes.

žThus, the number of chiasmata is roughly proportional to chromosome length.

Recombination frequency

žLet’s assume that genes A and B are on different chromosomes and that an AABB individual is crossed to an aabb individual.

žFrom this cross the AaBb offspring are then testcrossed to the double recessive parent (aabb).

žBecause the A and B genes assort independently, the F2 will consist of two classes (AaBb and aabb) that are phenotypically like the parents in the original cross and two classes (Aabb and aaBb) that are phenotypically recombinant.

žFurthermore, each F2 class will occur with a frequency of 25 percent. Thus, the total frequency of recombinant progeny from a testcross involving two genes on different chromosomes will be 50 percent.

žA frequency of recombination less than 50 percent implies that the genes are linked on the same chromosome.