UNIT 4: CARBOHYDRATES, LIPIDS, VITAMINS Experiment 1: Qualitative Tests for Carbohydrates and Lipids Determination of Cholesterol by Cholesterol Oxidase

I. Background: Carbohydrates: The word carbohydrate entered the vocabulary of chemistry many years ago when people thought the empirical formula for the most carbohydrates, CH 2O, indicated they were hydrates of carbon, in which water molecules were associated with carbon atoms in some way. Shortly thereafter it was shown that these compounds were not hydrates at all, but rather aldehydes and ketones that contain several hydroxyl groups per molecule. Today, a carbohydrate is considered to be a polyhydroxyaldehyde or a polyhyroxyketone, or another compound that yields these upon hydrolysis (the reaction with water in the presence of acid). Carbohydrates are the principal constituents of plants, comprising 60to 90 percent of their dry weight. Plants use carbohydrates as an energy source, an energy storage medium, and as structural material. The building block of starch and cellulose is glucose, which is produced in photosynthesis. Carbohydrates are divided into three major classes, based on their behavior when exposed to acid hydrolysis: 1. Monosaccharides: Not broken down into simpler carbohydrates by acid hydrolysis. These are often called the simple sugars. 2. Disaccharides: Yield two monosaccharides upon acid hydrolysis. Sucrose, the most abundant disaccharide yields glucose and fructose. 3. Polysaccharides: Yield more than two monosaccharides upon acid hydrolysis Monosaccharides are the simplest carbohydrates with cyclic structure. Three common monosaccharides are glucose, galactose, and fructose. Disaccharides are composed of two monosaccharides joined by an ether (-C-O-C-) linkage. Disaccharides are formed in nature from two monosaccharides, which are joined with the elimination of one water molecule. In solution, the cyclic molecule exists in equilibrium with the open-chain structure, which contains either an aldehyde or ketone functional group. Of the three common disaccharides, the unit of lactose and maltose is able to open, forming an aldehyde group. Sucrose, because of the manner in which the fructose and glucose cyclic units are joined cannot undergo such a ring opening. Polysaccharides are complex carbohydrates made up of many monosaccharide units joined together by ether linkages to form a long polymeric structure. Starch and glycogen are the most important polysaccharides, and they are polymers of repeating glucose units. Carbohydrates may also be classified as reducing sugars. Reducing sugars have a free aldehyde group or potential aldehyde group, such as hemiacetal, or keto and hemiketal groups that are isomerized to an aldehyde under alkaline conditions. These groups may be oxidized, under alkaline conditions, by certain metal ions that become reduced. The reduction of the metal ion indicates that the sugar is a reducing carbohydrate. A commonly used ion is Cu+2.

Cholesterol: One of the best known and best studied of the lipid groups is the steroids class. Cholesterol, the well-known steroid, is amphiphilic with a polar head (the OH group) and an extensive nonpolar region (the fused rings and hydrocarbon tails). Indeed, cholesterol and some of its derivatives accompany the glycerophospholipids and sphingolipids in biological membranes. The most chemically reactive portion of the cholesterol structure is the hydroxyl group. Under physiological conditions, it is common for a fatty acid to be esterified at this position. Cholesterol and its ester derivatives are abundant in plasma proteins called lipoproteins whose function it is to transport the cholesterol to peripheral tissue for use in construction of membranes and as a biosynthetic precursor for steroid hormones and other biologically active products. Measurement of serum cholesterol levels can serve as an indicator of liver function, biliary function, intestinal absorption, and propensity toward coronary artery disease, thyroid function and adrenal disease. Cholesterol levels are important in the diagnosis and classification of hyperlipoproteinaemias. Stress, age, gender, hormonal balance and pregnancy affect normal cholesterol levels. II. Objectives: 1. To measure plasma cholesterol levels by using cholesterol oxidase. 2. To test different carbohydrates and lipids qualitatively. 3. To test milk qualitatively for its carbohydrate and lipid groups III. Procedure: THE MOLISH TEST: It is a general test for the presence of most carbohydrates, such as mono, oligo, and polysaccharides. The test is negative for sugar alcohols (alditols) and 2-deoxy-2amino sugars. The concentrated sulfuric acid hydrolyzes any glycosidic bonds present to monosaccharides, which are then dehydrated by the concentrated acid to yield furfural or one of its derivatives (e.g., hydroxymethylfurfural). The furfural compounds condense with sulfonated -naphthol to give a purple complex, which is characteristic of carbohydrates. The limit of detection is 10 g/mL. Pentoses give furfural; hexoses give 5-hydroxymethylfurfural; hexuronic acids decarboxylate to give furfural; and 6deoxyhexoses give 5-methylfurfural. The exact structure of the condensation products with sulfonated -naphthol (the purple complex) is unknown. Assay Procedure: 1. Prepare and label 6 test tubes as directed below: 1 ml of distilled water (to control) 1 ml of glucose solution 1 ml of raffinose solution 1 ml of lactose solution 1 ml of starch solution 1 ml of milk

2. Add 2 drops of the -naphthol reagent to each tube. 3. Then pour 1 ml of concentrated sulfuric acid down the side of the tube to form a layer on the bottom. 4. If carbohydrate is present, a purple complex will be formed at the interface between the two solutions. BENEDICTS REDUCING TEST : This test is used to detect the presence of reducing sugars. The Benedict test solution contains copper(II)ion, Cu2+, in a soluble complex, and sodium hydroxide. This alkaline solution can oxidize the aldehyde group to the carboxylic acid group, as copper(II) ion is reduced to copper(I) oxide, a brick-red precipitate. The formation of the highly colored precipitate is visual evidence of the presence of a reducing sugar. Assay Procedure: 1. Prepare and label 6 boiling test tubes that contain 200 L of distilled water, glucose, raffinose, lactose, starch, or milk. 2. Add 1 mL of Benedicts reagent into each tube. 3. Place the tubes into a boiling water bath for 5 minutes. 4. Check the color changes in the tubes. THE IODINE TEST: This test detects the presence of polysaccharides. Polysaccharides combine with iodine to form a blue, red violet, or purple color as the signal of a positive test. Iodine is adsorbed onto the surface of the polysaccharide, forming a colored compound. Assay Procedure: 1. Prepare and label 6 test tubes like in the Molish Test. 2. Add one drop of iodine solution to each tube and shake to mix. 3. Observe the control sample. If none of the samples has a color different than the control, add 10 drops of distilled water to each test tube and one more drop of the iodine solution. 4. Record your observations. THE GREASE SPOT TEST: This is a simple test to show the presence of triglycerides. An acetone solution of the test sample is poured onto a piece of filter paper. After the acetone evaporates, a translucent grease spot remains behind as evidence of the triglyceride. Assay Procedure: 1. Place a small drop of vegetable oil on a piece of filter paper. 2. Mix 1 mL of milk with 2 mL acetone. Place a few drops of the solution on the center of another filter paper. 3. Hold the papers up to the light. Record your observations and compare.

THE LIEBERMANN-BURCHARD TEST: This test detects the steroids. Acetic anhydride can react with the C3 hydroxyl group of cholesterol and related steroid is the presence of strong acids to form a bluegreen complex. The reaction must be carried out in the absence of water. Assay Procedure: 1. Prepare two test tubes with 1 ml of cholesterol and ergosterol solutions in chloroform. Use only glass pipets. 2. Add 5-6 drops of acetic anhydride to each tube with gentle mixing. 3. Cautiously add 0.5 mL of concentrated sulfuric acid down the side of the tube without mixing. 4. Record your observation immediately. 5. The appearance of a blue-green color is a positive test for cholesterol. Ergosterol quickly develops a red color. DETERMINATION OF CHOLESTEROL BY CHOLESTEROL OXIDASE: Cholesterol is oxidized to cholestenone and hydrogen peroxide by cholesterol oxidase. In the presence of peroxidase, the H2O2 reacts with 2-amino-antipyrine and phenol to form a colored complex that can be quantitated by measuring the absorbance at 500 nm.
cholesterol oxidase

Cholesterol + O2 cholestenone + H2O2

peroxidase

H2O2 + phenol + 2-aminoantipyrine colored complex + H2O Since only free cholesterol is a substrate for cholesterol oxidase, the determination of total cholesterol (free cholesterol + cholesterol esters) requires that the sample first be treated with cholesterol esterase, which catalyzes the hydrolysis of cholesterol esters to free cholesterol. Assay Procedure: 1. The cholesterol stock solution concentration is 50 mg/ml. 2. Make appropriate dilutions to obtain 40, 25, 10, and 5 mg/ml cholesterol solutions for your standard curve. 3. Take 30 L of the standard solutions and add 3 mL of reagent. 4. Take 30 L of plasma and add 3 mL of reagent. 5. Take 30 L of unknown cholesterol sample and add 3 mL of reagent. 6. Parafilm and incubate the tubes for 15 min at room temperature. 7. Read the absorbances at 500 nm.

IV. Reagents: 1. 5%(w/v) -naphthol in ethanol. 2. Concentrated sulfuric acid 3. Benedicts Reagent: 173 g sodium citrate and 100 g of sodium carbonate in 800 mL of warm distilled water and 17.3 g of copper sulfate in 200 mL of distilled water. The copper sulfate solution is slowly added to the citratecarbonate solution with stirring. 4. Acetic anhydride 5. Cholesterol and ergosterol solutions in chloroform. 6. Cholesterol stock solution in water 7. Cholesterol oxidase reagent. 8. 1 % of glucose, raffinose, lactose, starch solutions 9. Milk, vegetable oil. V. Observations and Calculations: 1. Draw or describe your observations for all qualitative tests. Discuss your results in the discussion section of your report. 2. Construct a standard curve and calculate the cholesterol concentrations in unknown sample and plasma. VI. Questions: 1. What was your negative control in Liebermann-Burchard Test? 2. Show the reactions between acetic anhydride and cholesterol/ergosterol. 3. Draw the structure of cholesterol and explain the chemical basis of cholesterol oxidase test. 4. What do LDL and HDL stand for? Explain their importance in biological systems.

UNIT 4 Exp1 DATA SHEET: