Carbohydrates PDF

CARBOHYDRATES SHARI BABU DEPT. OF PHYSIOLOGICAL SCIENCES SCHOOL OF MEDICINE [email protected]  INTRODUCTION Carbohydrates are widely distributed in plants and animals. They are the most abundant biomolecules on the Earth. Carbohydrates are polyhydroxy aldehyde...

CARBOHYDRATES SHARI BABU DEPT. OF PHYSIOLOGICAL SCIENCES SCHOOL OF MEDICINE [email protected]  INTRODUCTION Carbohydrates are widely distributed in plants and animals. They are the most abundant biomolecules on the Earth. Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield such compounds on hydrolysis. Many, but not all, carbohydrates have the empirical formula (CH2O)n. Carbohydrates can be classified based on their size: monosaccharides, disaccharides, oligosaccharides, and polysaccharides.  FUNCTIONS OF CARBOHYDRATES Certain carbohydrates (sugar and starch) form dietary staple, and the oxidation of carbohydrates is the central energy-yielding pathway. Insoluble carbohydrate polymers serve as structural and protective elements in the cell walls of bacteria and plants and in the connective tissues of animals. Other carbohydrate polymers lubricate skeletal joints. Specific carbohydrate containing molecules act in cell-cell recognition and adhesion, cell migration during development, blood clotting, the immune response, and wound healing.  MONOSACCHARIDES They are simple sugars, consist of a single polyhydroxy aldehyde or ketone unit. The most abundant monosaccharide in nature is D-glucose, sometimes referred to as dextrose. They may be classified as trioses, tetroses, pentoses, hexoses, or heptoses, depending upon the number of carbon atoms. As aldoses or ketoses depending upon whether they have an aldehyde or ketone group. H O C CH2OH H C OH C O HO C H HO C H H C OH H C OH H C OH H C OH CH2OH CH2OH D-glucose D-fructose  MONOSACCHARIDES The simplest monosaccharides of biological significance are trioses glyceraldehyde and dihydroxyacetone. Monosaccharides of more than five carbons tend to have cyclic structures. Monosaccharides are usually colorless, water-soluble, crystalline solids. Some monosaccharides have a sweet taste. ALDOSES KETOSES ISOMERS Compounds that have the same chemical formula but have different structures are called isomers. – For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, C6H12O6. EPIMERS Carbohydrate isomers that differ in configuration around only one specific carbon atom are defined as epimers of each other. – For example, glucose and galactose epimers are C-4. Glucose and mannose are C-2 epimers. ENANTIOMERS Another type of isomerism involves a pair of structures that are mirror images of each other. The mirror images are called enantiomers, and they are designated as a D- and an L-sugar. In the D-isomer, the –OH group on the asymmetric carbon farthest from the carbonyl carbon is on the right, whereas in the L-isomer it is on the left. Most of the sugars in humans are D-sugars. CYCLIZATION OF MONOSACCHARIDES Monosaccharides with five or more carbons exists predominantly in a ring (cyclic) form, in which the aldehyde or keto group react with a -OH group on the same sugar. Reaction between alcohols and aldehydes or ketones form derivatives called hemiacetals or hemiketals, respectively. D-glucose forms hemiacetal in which the hydroxyl group at C-5 reacts with the aldehyde group at C-1, forming a six-membered cyclic structure called pyranose (glucopyranose). Aldohexoses can also exist in cyclic forms having five membered rings called furanoses. – The six-membered aldopyranose ring is much more stable than the aldofuranose ring. In ketohexoses, the hydroxyl group at C-5 reacts with the keto group at C-2, forming a furanose ring containing a hemiketal linkage. – D-Fructose readily forms the furanose ring  Isomeric forms of monosaccharides that differ only in their configuration about the hemiacetal or hemiketal (anomeric) carbon atom are called anomers. – Two forms α and β anomers.  SUGAR DERIVATIVES Organisms contain different type of sugar derivatives. Majority of sugar derivatives are derivatives of hexose sugar that have been modified. Sugar derivatives are formed either by: – Substitution of hydroxyl group by another group – Oxidation to form carboxyl group 1. ALDONIC ACIDS Aldonic acids are formed by the oxidation of the aldehyde group at C1 position to a carboxylic group. For example, glucose is oxidized to gluconic acid. These acids have a broad range of applications in the food industry as gelling and solubilizing agents, in cosmetics as antioxidants. 2. URONIC ACID Uronic acids are formed by the oxidation of the hydroxyl group at C6 position to a carboxylic group. Uronic acids can assume pyranose, furanose or linear conformation. For example, glucose is converted to glucuronic acid. Glycosides derived from D-glucuronic acid and steroids appear in the urine of animals as normal metabolic products. Foreign toxic substances are frequently converted in the liver to glucuronides before excretion in the urine (conjugation). D-Glucuronic acid also is a major component of connective tissue polysaccharides. 3. ALDARIC ACIDS Aldaric acids are formed when both the aldehyde group at C1 and the hydroxyl group at C6 are oxidized to carboxyl groups. Aldaric acids do not form cyclic structures. For example, glucose is converted to glucaric acid. Glucaric acid acts as a chelating agent that binds to calcium and magnesium ions 4. ALDITOLS Alditols are formed by the reduction of aldose to polyhydroxy alcohols. For example, glucose is reduced to sorbitol. They are used widely in the food industry as thickeners and sweeteners. Used as a sugar substitutes for diabetic patients 5. DEOXY SUGARS Monosaccharides in which a hydroxyl group is reduced to a hydrogen atom is called deoxy sugars. For example, β-D-ribose is reduced to β-D-2 deoxyribose. They are important for the formation of purine and pyrimidine nucleotides. 6. AMINO SUGARS In amino sugars, the hydroxyl group at C2 position is replaced with an amino group. For example, glucose is converted to glucosamine. The amino group is nearly always condensed with acetate to form N- acetylglucosamine, which is part of many structural polymers, including those of the bacterial cell wall. 7. SIALIC ACIDS These are nine-carbon sugar derivatives that are usually present as terminal residues of glycans and glycoconjugates. An important example is N-acetylneuraminic acid. – N-acetylmannosamine reacts with phosphoenolpyruvate to form N- acetylneuraminic acid. N-acetylneuraminic acid and its derivatives are collectively called Sialic acids. They play an important role in cellular communication, and in stabilizing cellular membranes. They help in binding and transporting ions and drugs. Stabilizing the formation of proteins such as enzymes. They protect molecules and cells from attack by proteases or glycosidases.  DISACCHARIDES Disaccharides are condensation products of two monosaccharide units. Examples are maltose, lactose and sucrose. Reducing sugars have a free aldehyde or keto group. Two monosaccharides joined covalently by glycosidic bond.  DISACCHARIDES Disaccharides can be hydrolyzed to yield their free monosaccharide components by boiling with dilute acid. The disaccharide maltose contains two D-glucose residues. It is hydrolyzed by maltase. The disaccharide lactose, which yields D-galactose and D-glucose on hydrolysis, occurs naturally only in milk. It is hydrolyzed by lactase. - In lactase deficiency, malabsorption leads to diarrhoea and flatulence. - Lactose accumulates in the lumen of the small intestine because there is no mechanism for the uptake of the disaccharide. - Osmotic effects of unabsorbed lactose leads to influx of fluid into the small intestine Sucrose (table sugar) is a disaccharide of glucose and fructose. It is formed by plants but not by animals.  POLYSACCHARIDES Most carbohydrates found in nature occur as polysaccharides, also called glycans. Homopolysaccharides contain only a single type of monomer; heteropolysaccharides contain two or more different kinds. Some homopolysaccharides serve as storage forms of monosaccharides that are used as fuels; starch and glycogen are homopolysaccharides of this type. Other homopolysaccharides (cellulose and chitin, for example) serve as structural elements in plant cell walls and animal exoskeletons. Heteropolysaccharides provide extracellular support for organisms of all kingdoms.  HOMOPOLYSACCHARIDES The most important storage polysaccharides are starch in plant cells and glycogen in animal cells. Both polysaccharides occur intracellular as large clusters or granules. Starch and glycogen molecules are heavily hydrated, because they have many exposed hydroxyl groups available to hydrogen-bond with water. STARCH Found in in tubers, such as potatoes, and in seeds. Made up of two types of glucose polymer, amylose and amylopectin. Amylose consists of long, unbranched chains of D-glucose residues connected by (α1  4) linkages. Amylopectin is highly branched. The successive glucose residues joined by (α1  4); the branch points (occurring every 24 to 30 residues) formed by (α1  6) linkages. Both amylose and amylopectin are hydrolyzed by α –amylase. – α–amylase hydrolyzes internal α1→4 linkages to yield maltose (2 glucose residues in α1→4 linkage), maltotriose (3 glucose residues in α1→4 linkages), and α-dextrin (several glucose units joined by α1→6 linkage and α1→4 linkages). STARCH AMYLOSE AMYLOPECTIN GLYCOGEN Glycogen is the main storage polysaccharide in animal cells. It is a polymer of (α1  4)-linked subunits of glucose, with (α1  6) at branch points. – Glycogen is more extensively branched (on average, every 8 to 12 residues) and more compact than starch. Glycogen is most abundant in the liver and in skeletal muscle. In hepatocytes, glycogen is found in large granules. – Glycogen granules contain the enzymes responsible for the synthesis and degradation of glycogen.  HETROPOLYSACCHARIDES PEPTIDOGLYCANS The rigid component of bacterial cell walls is a heteropolymer of alternating (β1  4) linked N-acetylglucosamine and N-acetylmuramic acid residues. The linear polysaccharides lie side by side in the cell wall, cross-linked by short peptides.  The cross-links hold the polysaccharide chains into a strong sheath that envelopes the entire cell and prevents cellular swelling and lysis due to osmotic entry of water. The enzyme lysozyme kills bacteria by hydrolyzing the (β1  4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid. Lysozyme is present in tears, presumably as a defense against bacterial infections of the eye. It is also produced by certain bacterial viruses to ensure their release from the host bacterial cell. Penicillin and related antibiotics kill bacteria by preventing synthesis of the cross-links, leaving the cell wall too weak to resist osmotic lysis. GLYCOSAMINOGLYCANS Glycosaminoglycans, are a family of linear polymers composed of repeating disaccharide units. One of the two monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine; the other is in most cases a uronic acid. The extracellular matrix is composed of an interlocking meshwork of heteropolysaccharides (Glycosaminoglycans) and fibrous proteins such as collagen, elastin, fibronectin etc. The glycosaminoglycan, hyaluronic acid contains alternating residues of D- glucuronic acid and N-acetylglucosamine.  Hyaluronates are highly viscous solutions that serve as lubricants in the synovial fluid of joints and give the vitreous humor of the vertebrate eye its jelly-like consistency. – Hyaluronate is also an essential component of the extracellular matrix of cartilage and tendons. Chondroitin sulfate contributes to the tensile strength of cartilage, tendons, ligaments, and the walls of the aorta. – Formed of alternating N-acetylgalactosamine and glucuronic acid Dermatan sulfate contributes to the pliability of skin and is also present in blood vessels and heart valves. – Formed of alternating N-acetylgalactosamine and iduronic acid.  Keratan sulfates are present in cornea, cartilage, bone, and a variety of structures formed of dead cells: horn, hair, hoofs, nails, and claws. – Formed of alternating N-acetylglucosamine and galactose Heparin is a natural anticoagulant made in mast cells (a type of leukocyte) and released into the blood, where it inhibits blood coagulation by binding to the protein antithrombin. – Formed of alternating N-acetylglucosamine and either glucuronic acid or iduronic acid. Glycoconjugates: Proteoglycans, Glycoproteins, and Glycolipids These are carbohydrates that are covalently linked to other biomolecules like proteins and lipids. Proteoglycans are macromolecules on the cell surface or extracellular matrix in which one or more glycosaminoglycan chains are joined covalently to a membrane protein or a secreted protein. – The glycosaminoglycan commonly forms the greater part of the proteoglycan molecule. – It is often the main site of biological activity. Proteoglycans are major components of connective tissue such as cartilage.  Glycoproteins have one or several oligosaccharides of varying complexity joined covalently to a protein. They are found on the outer face of the plasma membrane, in the extracellular matrix, and in the blood. They are found in specific organelles such as Golgi complexes, secretory granules, and lysosomes. The oligosaccharide portions of glycoproteins forms highly specific sites for recognition and high-affinity binding by other proteins. Many of the proteins secreted by eukaryotic cells are glycoproteins; e.g. antibodies and hormones like luteinizing hormone, follicle stimulating hormone etc.  Glycolipids are membrane lipids in which the hydrophilic head groups are oligosaccharides. – Gangliosides are membrane lipids of eukaryotic cells made up of sialic acid and glycosphingolipid. – The oligosaccharide groups on gangliosides help in cell recognition and cell-to- cell communication. – They also act as specific receptors. Lipopolysaccharides are the dominant surface feature of the outer membrane of gram-negative bacteria. – Lipopolysaccharides are important targets of the antibodies produced in response to bacterial infection and are important determinants of bacterial strain. QUALITATIVE & QUANTITATIVE ANALYSIS QUALITATIVE ANALYSIS Qualitative tests are used to detect and identify the type of carbohydrates present in a given sample. The qualitative analysis of carbohydrate is based on the reaction between the test sample and reagent. The reaction of the test sample with the chemical reagent gives a significant color, through which the presence or absence, and the type of carbohydrates can be detected. Molisch’s Test It is the most common method for the detection of carbohydrates. The test uses Molisch’s reagent (contains α-naphthol in 95% alcohol) and concentrated H2SO4. It detects all types of carbohydrates. Principle: The principle is based upon dehydration reaction. The carbohydrates in the sample get dehydrated to form an aldehyde by the addition of conc. H2SO4. The aldehyde formed is either furfural (produced by the dehydration of pentoses) or hydroxymethylfurfural (produced by the dehydration of hexoses). The α-naphthol in the Molisch’s reagent condenses with the aldehyde and develops a purple ring at the junction of the H2SO4 and test sample. http://dept.harpercollege.edu/ Benedict’s Test It is used for the identification of reducing sugars. Reducing sugar consists of a free aldehyde or ketone group. The test uses Benedict’s solution, which contains copper sulphate, sodium citrate, sodium carbonate with a pH of 10.5. Principle: The reducing sugar will reduce into the enediols by reacting with alkaline reagent. The reducing sugar gives green to brick red color precipitate depending upon the sugar concentration. The color change is due to the reduction reaction of copper (II) to copper (I) in the solution that develops a red colored precipitate. https://byjus.com/ Fehling Test This test detects reducing aldehyde sugars. It makes the use of Fehling’s solution A and B as a chemical reagent. – Fehling’s solution A contains copper (II) sulphate. – Fehling’s solution B contains potassium sodium tartrate and sodium hydroxide. Principle: In the Fehling test, a reduction reaction occurs between the aldehyde group of the reducing sugar and the alkaline cupric hydroxide that later reduces into cuprous oxide. This cuprous oxide gives a brick red colored precipitate in the solution. Common use of Fehling test is to determine whether a carbonyl group is an aldehyde or a ketone. https://byjus.com/ Barfoed’s Test This test is used to distinguish reducing monosaccharide from disaccharides. Barfoed’s test makes the use of Barfoed’s solution that contains copper acetate in the dilute acetic acid with a pH of 4.6. Principle: The reducing monosaccharide is oxidized by the copper ion in the solution to form copper (I) oxide, which results in the formation of a red colored precipitate. Reducing monosaccharides react with Bedford’s reagent much faster than disaccharides and produce a red precipitate within three minutes. Disaccharide sugars as they are weaker reducing agents, react at a slower rate. http://dept.harpercollege.edu/ Bial’s Test It is most commonly used for the detection of pentose sugars. Bial’s test makes the use of Bial’s solution that contains orcinol, hydrochloric acid and ferric chloride. Principle: Here, the pentoses react with the Bial’s reagent and is dehydrated into furfural derivatives due to dehydration by HCl. Then, orcinol and furfural condense in ferric ion presence and develop a bluish colored compound. A green, red, or brown color indicates hexoses, and disaccharides are yellow. http://dept.harpercollege.edu/ Seliwanoff’s Test It is most commonly used for the detection of ketoses. Seliwanoff’s test uses Seliwanoff’s reagent that contains resorcinol in HCl. Principle: In this test, ketoses react with the HCl of the Seliwanoff’s reagent and yield furfural derivatives due to dehydration. Then, resorcinol and furfural react to give a deep red color to the solution. Aldohexoses react to form the same product, but do so more slowly. http://dept.harpercollege.edu/ Iodine Test It is widely used for the detection of starch in the solution. In this test, iodine acts as an indicator. Principle: Iodine solution reacts with the α-amylose. This reaction between amylose and iodine results in a complex, which gives a blue-black color to the solution. QUANTITATIVE ANALYSIS Quantitative analysis is carried out to determine the concentration of a particular compound in a given sample. The quantitative analysis of carbohydrates, specifically glucose is carried out by colorimetric method using a spectrophotometer. Quantitative analysis of glucose can be carried out by the following methods: – Hexokinase/Glucose-6-phosphate Dehydrogenase method – Glucose oxidase-Peroxidase method – O-toluidine method – Copper reduction method The advantages of using enzyme-based methods compared to chemically based methods are: – Have higher specificity, – Occur at lower temperatures, and – In many cases use less hazardous reagents Hexokinase/Glucose-6-phosphate Dehydrogenase method Hexokinase catalyzes the formation of glucose 6-phosphate (G6P) from glucose. G6P is then oxidized to gluconolactone 6-phosphate by G6P dehydrogenase (G6PD) while NADP+ is reduced to NADPH. The concentration G6P, and by extension the concentration of glucose can be determined by measuring NADPH absorbance using a spectrophoto- meter at 340 nm. This method is performed using a single premixed reagent solution containing the enzymes. Glucose oxidase-Peroxidase method In this method, glucose is oxidized by glucose-oxidase (GOD) in the presence of atmospheric oxygen to gluconolactone and hydrogen peroxide. The hydrogen peroxide is then oxidized by peroxidase (POD) in the presence of 4-aminophenazone and phenol to form the red dye (4-(p- benzochinone-monoimino)-phenazone), which is quantitated spectrophotometrically at 505 nm. O-toluidine method This is a chemical method for the determination of glucose in sample. It is based on the reaction of glucose in hot acidic solution with the aromatic amine, o-toluidine, to form a Schiff base whose absorbance is measured at 630 nm. Disadvantage of this method is that sugars like galactose and mannose will also react nonspecifically to give a colored product, – the reaction must be carried out at elevated temperatures (80–100 °C), and – the reagent is hazardous. However, it may still be of value in developing countries because it is technically simple and inexpensive. Copper reduction method (modified Folin-Wu method) When glucose is treated with an alkaline copper solution there is reduction of copper into insoluble cuprous oxide. The cuprous oxide formed is allowed to react with phosphomolybdate to form molybdenum, a blue colored complex which can be read colorimetrically at 420 nm. The reaction depends on temperature, duration of heating, a degree of alkalinity.