BIOCHEMISTRY AND HUMAN NUTRITION

Module 4. Human nutrition

Lesson 34
ESTIMATION OF VITAMIN C AND CHOLESTROL

34.1 Introduction

• Vitamin C also called ascorbic acid is a water soluble vitamin, present in nearly all fresh fruits and vegetables.
• It is Heat labile i.e. destroyed on heating. It is present in all higher plants.
• Human beings cannot synthesize this vitamin and hence require it in their diet. Daily requirement is 40 mg. Inability to synthesize vitamin C is because they genetically lack the ability to synthesize the enzyme
• Gluconolactone oxidase is required for the formation of vitamin C.
• Vitamin C cannot be stored in the body; excessive intake causes it to be secreted in the urine.
• The vitamin is neither present nor is required by microorganisms.
• Starting compound for the synthesis of vitamin C is D- Glucose. The biologically active form is L-ascorbic acid. D- ascorbic acid does not possess any antiscorbutic activity.
• Ascorbic acid in solution is rapidly oxidized to dehydroascobic acid. The oxidation is catalysed by copper and silver ions. The oxidation is faster at higher temperatures e.g. during cooking of foods.

34.2 Significance

• Regular intake of C is required for healthy bones and teeth. Vitamin C is essential for the growth of subcutaneous tissue, cartilage, bone and teeth.
• In the absence of Vitamin C, the collagen that is formed is defective and weak. This is because vitamin C is required for activating the enzyme, Prolyl hydroxylase, that promotes the hydroxylation step in the formation of hydroxyproline, an integral constituent of collagen. Hydroxyproline is found only in collagen and no other animal protein.
• Collagen is a fibrous protein present in all multicellular organisms and is the most abundant protein in mammals, constituting a quarter of the total. Collagen is the major fibrous element of skin, bone, tendon, cartilage, blood vessels and teeth. It is present to some extent in nearly all organs and serves to hold cells together in discrete units.
• Deficiency of vitamin C causes a disease called Scurvy, which has got the following effects:

• Failure of wounds to heal.
• Bone growth stops. No new bone formation occurs. In already formed bone, fractures if any, do not heal.
• Blood vessels become extremely fragile and rupture easily, especially capillaries.
• In extreme scurvy, muscle cells fragment, lesion of gums with loosening of teeth occurs. Severe hemmorages can eventually lead to death.

34.3 Principle

• Ascorbic acid reduces the indicator dye to a colorless solution. At the end point of titrating an ascorbic acid containing sample with dye, excess unreduced dye is rose- pink color in acid solution. The titer of dye can be determined using standard ascorbic acid solution. Food samples in solution then can be titrated with the dye and the volume of titration used to calculate the ascorbic acid content.
• Ascorbic acid reduces the dye 2, 6 – dichlorophenol indophenols to colourless, and itself gets oxidized to dehydroascorbic acid. Though the dye is a blue coloured compound, the end point is the appearance of pink colour for 5-10 seconds. The dye is pink in acid medium. A mixture of metaphosphoric and acetic acid is used as the titrating medium.

34.3.1 Reagents

1. Metaphosphoric acetic acid mixture: Dissolve 15 g of metaphosphoric acid in a mixture containing 40 ml glacial acetic acid and 450 ml distilled water. Filter and store in refrigerator. Stable for 10 days.
2. Dye solution: Dissolve 42 mg sodium bicarbonate and 50 mg 2, 6 – dichlorophenol indophenol dye in 50 ml of distilled water. Make up the volume to 200 ml. Filter and store in the refrigerator
3. Standard Stock solution: 1 mg/ ml. Dissolve 100 mg of ascorbic acid in 100 ml of 3% metaphosphoric acetic acid mixture.
4. Working Standard solution: 0.1mg/ ml. Dilute 10 ml of the stock to 100 ml with 3% metaphosphoric acetic acid mixture.

34.3.2 Protocol

• Pipette out 5 ml of the working standard solution into a 100 ml conical flask. Add 10 ml of acetic metaphosphoric acid mixture and titrate with dye. End point is the appearance of pink colour which persists for 5-10 seconds.
• Extract the sample (0.5 – 5 g depending upon the nature of the sample) in acetic metaphosphoric acid mixture and make up to a known volume e.g. 100 ml. Filter. Take 5 ml of the filterate, add 10 ml of the acid mixture and titrate against dye.

34.3.3 Observations

Volume of dye used in titration of standard in ml
Volume of dye used in titration of sample in ml

34.3.4 Calculations

Express results as concentration of vitamin C per ml of extract and per gm of sample.

34.3.5 Notes

1. 4% oxalic acid may be used in place of metaphosphoric: acetic acid mixture. The mixture is used as a titrating medium. It not only reduces the pH, thereby stabilizing vitamin C, but also forms complexes with metallic ions e.g. copper, thereby preventing the catalytic oxidation of the vitamin.
2. The titration is conducted in the presence of acetic and metaphosphoric acids in order to inhibit aerobic oxidation catalysed by certain metallic ions, to inactive the enzyme, ascorbic acid oxidase, to precipitate proteins and liberate protein bound ascorbic acid.
3. Certain batches of metaphosphoric acid may have impurities that reduce the dye. Hence, reagents used should always be checked to guard against this possibility. A reagent blank with addition of dye should always be run along with sample.

34.4 Estimation of Cholesterol by Colorimetric Method

34.4.1 Cholesterol

• Cholesterol is a member of a large group of substances called steroids, which include vitamin D and a number of steroid hormones, among them are the sex hormones of higher animals. (Fig 34.1)
• Cholesterol is an essential component of cell membranes, brain and nerve cells, and bile, which helps the body absorb fats and fat soluble vitamins.
• Normal level varies with age, diet and from one location to another, however the average is 150-250 mg/dl blood.
• Cholesterol may be elevated slightly in pregnancy

34.4.2 Principle

The reaction depend on the colorimetric determination of a green colored compound resulting from the reaction of cholesterol with acetic anhydride-sulphuric acid (strong dehydrating and oxidizing agent) the absorbance of the green compound is then measured at 610 nm.

34.4.3 Material

• Cholesterol reagent (Acetic anhydride + acetic acid)
• Sulphuric acid 95-97%
• Standard cholesterol (300 mgl /dl)
• Samples
• Test tupes
• Pipettes
• Cuvette
• Spectrophotometer
• Water path
  

Fig. 34.1 Chloestrol