CCH-101 [Dr. H.A.

Khan]

CLINCIAL CHEMISTRY CCH-101 Class Notes

Dr. Haseeb Ahmad Khan Department of Biochemistry College of Science King Saud University

Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

LIPIDS STRUCTURE
Definition
Lipids are organic compounds that are insoluble in water and soluble in nonpolar organic solvents such as ether, acetone, chloroform, carbon tetrachloride etc. The biological importance of lipids is summarized below: 1. Lipids are most concentrated source of energy (9 kcal/g). 2. Lipids provide essential fatty acids. 3. Lipids are structural components of cell membrane. 4. Lipids supply fat soluble vitamins (A, D, E and K). 5. Lipids act as insulating material.

Classification: Lipids are divided into 3 categories: simple, complex and derived lipids.
Simple Lipids: Simple lipids are esters of fatty acids. The hydrolysis of simple lipids gives fatty acids and alcohol. hydrolysis Simple lipid + H2O fatty acids + alcohol

If the hydrolysis of a simple lipid yields 3 fatty acids and glycerol, the simple lipid is called fat or oil (fats are solid and oils are liquid at room temperature). If the hydrolysis of a simple lipid yields a fatty acid and a high molecular mass monohydric alcohol, the simple lipid is called wax.

Complex Lipids: The hydrolysis of complex lipids yields one or more fatty acids, an alcohol and some other type of compound. Phospholipids and glycolipids belong to the category of complex lipids. hydrolysis Phospholipid + H2O fatty acid + alcohol + phosphoric acid + a nitrogen compound

Phospholipids are further divided into (1) phosphoglycerides, in which the alcohol is glycerol and (2) phosphosphingosides, in which the alcohol is sphingosine.

Glycolipid + H2O

fatty acid + a carbohydrate + sphingosine (N containing alcohol)

Other complex lipids include sulfolipids, aminolipids and lipoproteins.

Derived Lipids: They are produced when simple and complex lipids undergo hydrolysis. They include ketone bodies, steroids, prostaglandins, and lipid-soluble vitamins.
Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

Fatty Acids
Fatty acids are straight chain organic acids. Fatty acids found in natural fats usually contain an even number of carbon atoms. Fatty acids are detergent like due to their amphipathic nature as they have nonpolar (CH3) and polar (COOH) ends.

Saturated and Unsaturated Fatty Acids: Saturated fatty acids contain only single bonds. Unsaturated fatty acids contain one or more double bonds. Unsaturated fatty acids have lower melting points than the corresponding saturated fatty acids. Greater the degree of unsaturation, the lower the melting point of a fatty acid. Longer the chain length, higher the melting point.

Name of fatty acid Stearic Acid Oleic Acid Linoleic Acid Linolenic Acid

Number of C atoms 18 18 18 18

Number of double bonds 0 1 2 3

Melting Point (C) 70 13 -5 -10

Most of the naturally occurring unsaturated fatty acids are of cis configuration (both H on the same side along a double bond). Hydrogenation produces a mixture of cis and trans isomers. Trans fatty acids are not good for health as they are responsible for lowering HDL (high density lipoproteins, good cholesterol) and raising LDL (low density lipoproteins, bad cholesterol). Examples of saturated fatty acids: Name Butyric Caproic Lauric Myristic Palmitic Stearic Formula C3H7COOH C5H11COOH C11H23COOH C13H27COOH C15H31COOH C17H35COOH Source Butter fat Butter fat Laurel Coconut oil Palm oil Peanut oil 3

Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

Examples of unsaturated fatty acids: Name Oleic Linoleic Linolenic Arachidonic Formula C17H33COOH (1 double bond) C17H31COOH (2 double bond) C17H29COOH (3 double bond) C19H31COOH (4 double bond) Source Olive oil Linseed oil Linseed oil Animal tissue, corn oil

Essential Fatty Acids (EFA): Two fatty acids are essential in humans: linoleic acid (precursor of prostaglandins) and linolenic acid. Arachidonic acid becomes essential if its precursor (linoleic acid) is missing in diet. A deficiency of EFA is characterized by scaly dermatitis, hair loss and poor wound healing.

Nomenclature of Fatty Acids: Carbon atoms are numbered from carboxylic carbon (carbon 1). The carbon atoms adjacent to the carboxylic carbon (2,3 and 4) are also known as , and respectively. The terminal methyl carbon is called carbon. 9 indicates a double bond on the 9th carbon from position. Whereas 9 indicates double bond between carbons 9 and 10 (from carboxylic group, C-1). The saturated fatty acids are suffixed with anoic (example, octanoic acid) and unsaturated fatty acids are suffixed with -enoic (example, octadecenoic acid).

CH3CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH = CH CH2 CH2 CH2 CH2 CH2 CH2 CH2COOH or CH3(CH2)7CH=CH(CH2)7COOH The above fatty acid is oleic acid (octadecenoic acid) and can be represented as 18:1:9 (18 carbon atoms, 1 double bond, at 9th carbon) or 9:C18:1 (1 double bond at 9) or 9: 18:1.

Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

Triacylglycerols
Triacylglycerols (triglycerides) are the esters of fatty acids and glycerol. They are principal storage fats in humans. Triglycerides are hydrolyzed to glycerol and fatty acids by lipase enzyme.

Phosphoglycerides
Phosphoglycerides are phosphate esters of diglycerides. Glycerol-3-phosphate is the structural backbone of phosphoglycerides. Two fatty acids are esterified to glycerol-3-phosphate to produce phosphatidic acid. Esterifying of choline with phosphoric acid portion of phosphatidic acid produces phosphatidylcholine (or lecithin). Lecithin plays an essential role in reducing surface tension in lung alveoli.

Phosphatidic Acid
Biochemistry lecture notes: Dr. Haseeb A. Khan

Phosphatidylcholine (Lecithin)

CCH-101 [Dr. H.A. Khan]

Sphingolipids
Sphingolipids contain fatty acids and sphingosine (in place of glycerol). Sphingosine is a long chain monounsaturated alcohol bound to ethanolamine. Ceramides, the simplest sphingolipids, consists of a fatty acid bound to sphingosine. Ceramides function principally as intermediates in synthesis of other sphingolipids. Sphingomyelins are produced by joining choline phosphate or ethanolamine phosphate to ceramides. Sphingomyelins are chiefly present in brain and nerve tissue.

Sphingosine Ceramide

Steroids
Steroids are high molecular mass tetracyclic compounds. The most common sterol is cholesterol which is found in animal fat but not in plants. Cholesterol is found in all animal tissues, particularly in brain and nervous tissue and blood. Deposition of cholesterol and triglycerides causes atherosclerosis. Ergosterol is a sterol similar to cholesterol. When ergoesterol is irradiated with UV light, it produces calciferol (vitamin D). Other steroids include bile salts, sex hormones and hormones of adrenal cortex.

Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

ANALYSIS OF LIPIDS
Iodine Number:
Iodine number indicates the degree of unsaturation of the lipid. Higher the iodine number, greater is the number of C=C double bonds or degree of unsaturation of fat. By definition the iodine value is expressed as the grams of iodine absorbed per 100g of lipid. Method- The lipid is weighed and dissolved in a suitable organic solvent, to which a known excess of iodine chloride is added. Some of the ICl reacts with the double bonds in the unsaturated lipids, while the rest remains: R-CH=CH-R + IClexcess R-CHI-CHCl-R + IClremaining The amount of ICl remaining is determined by adding excess potassium iodide to the solution to liberate iodine, and then titrating with a sodium thiosulfate (Na2S2O3) solution in the presence of starch to determine the concentration of iodine released: IClremaining + 2KI KCl + KI + I2 I2 + starch + 2Na2S2O3 (blue) 2NaI + starch + Na2S4O6 (colorless) While there is any I2 remaining in the solution it stays blue, but once all of the I2 has been converted to I it turns colorless. The concentration of C=C in the original sample can therefore be calculated by measuring the amount of sodium thiosulfate needed to complete the titration.

Saponification Value:
Saponification number is a measure of the average molecular weight of triacylglycerols. The smaller the saponification number the larger the average molecular weight of the triacylglycerol present. The saponification number is defined as the mg of KOH required to saponify 1 gram of fat. Saponification is the process of breaking down a neutral fat into glycerol and fatty acids by treatment with alkali: Triacylglycerol + 3 KOH Glycerol + 3 Fatty acid salts of potassium (Soap)

Method- The lipid is first extracted and then dissolved in an ethanol solution which contains a known excess of KOH. This solution is then heated so that the reaction goes to completion. The unreacted KOH is then determined by adding an indicator and titrating the sample with HCl. The saponification number is then calculated from knowledge of the weight of sample and the amount of KOH which reacted.
Biochemistry lecture notes: Dr. Haseeb A. Khan

CCH-101 [Dr. H.A. Khan]

Acid Value:
Acid value is defined as mg of KOH necessary to neutralize fatty acids present in 1g of lipid. Acid value is a measure of free acids present in fat. Acid value provides the information about the extent to which hydrolysis has liberated the fatty acids from their parent glyceride molecule. A rancid fat will have a higher acid value. Method- The lipids are extracted from the sample and then dissolved in an ethanol solution containing an indicator. This solution is then titrated with alkali (KOH) until a pinkish color appears. The acid value may be overestimated if other acid components are present in the system, e.g. amino acids or acid phosphates. The acid value is often a good measure of the break down of the triacylglycrols into free fatty acids, which has an adverse effect on the quality of many lipids.

Estimation of Blood Cholesterol:

Enzymatic Method: In this method, the enzyme cholesterol oxidase converts cholesterol into cholest-4-en-3-one and hydrogen peroxide. The cholest-4-en-3-one formed can be measured at 240 nm after extracting into isopropanol. Alternatively, H2O2 can be determined by Trinders reagent (4-aminophenazone + phenol + peroxidase). The enzyme determines only free cholesterol so for the determination of total cholesterol the ester cholesterol has to be hydrolyzed. This can be done by using ethanolic KOH or enzymatically by cholesterol ester hydrolase. cholesterol Cholesterol esters ester hydrolase cholesterol Cholesterol + O2 oxidase peroxidase H2O2 + 4-aminophenazone + phenol quinone-imine dye + H2O (colored) cholest-4-en-3-one + H2O2 cholesterol + fatty acids

Estimation of Serum Fatty Acids:

Serum is extracted with a chloroform-heptane-methanol mixture in presence of phosphate buffer. The extract is shaken with copper reagent (copper nitrate + triethanolamine + NaOH + NaCl, pH 8). The copper soaps remain in the upper organic layer and determined spectrophotometrically with diphenylcarbazide at 550 nm.
Biochemistry lecture notes: Dr. Haseeb A. Khan