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26 Estimation of High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) Cholesterol in Serum

26 Estimation of High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) Cholesterol in Serum

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Methods for Nutritional Quality Evaluation of Food Materials

flask containing glacial acetic acid, add 100 mg of uranyl acetate. Dissolve the

precipitate of ferric hydroxide and uranyl acetate in about 600 ml glacial acetic

acid. Make up the volume to 1.0 l with glacial acetic acid and mix well.

Ferrous acetate/sulphuric acid reagent: Weigh 100 mg of anhydrous ferrous

sulphate in 1 l volumetric flask, add 100 ml of glacial acetic acid and 100 ml

of concentration sulphuric acid. Mix thoroughly. Add 700 ml more of

concentrated sulphuric acid and mix. Cool to room temperature and make up

the volume. Mix thoroughly and store in a brown coloured bottle.

Sodium phosphotungstate reagent: Dissolve 40 g of phosphotungstic acid in 1 l

of solution (160 ml of 1 M sodium hydroxide + 840 ml of distilled water) to get

4% of final concentration.

2 M magnesium chloride solution: Dissolve 190 g of magnesium chloride in 1 l

of water.

Standard cholesterol: Dissolve 200 mg cholesterol in 100 ml glacial acetic acid.

Precipitating mixture: It is prepared fresh. Mix 1 part of reagent 4 with 4 parts of

reagent 3 (v/v).

HDL Cholesterol


1. To 1 ml of serum in a centrifuge tube add 0.1 ml of precipitating mixture. Allow

it to stand for 30 min. Centrifuge at 4,000 rpm for 30 min.

2. Separate the supernatant and to 0.2 ml supernatant add 5 ml of reagent 1.

3. Prepare a blank by taking 0.2 ml acetic acid and 5 ml of reagent 1.

4. Run a standard consisting of 0.1 ml of reagent 5, 0.1 ml of glacial acetic acid and

5 ml of reagent 1. Keep it for 10 min for complete precipitation and centrifuge.

5. In another set of clean and dry test tubes add 2 ml of reagent 2 and add to each

tube 3 ml of the supernatant from the above reaction mixture. Mix the contents,


6. Cool the tubes to room temperature and read the intensity at 560 nm against

blank. Calculate the amount of cholesterol by comparing the standing reading.

Dilution factor should be taken into account


HDL cholesterol in serum (mg/100 ml) =

Absorbance of test sample


Absorbance of standard

VLDL + LDL Cholesterol


1. To 1 ml of serum add 0.1 ml of precipitating mixture. Allow to stand for 20 min

at room temperature of complete precipitation. Centrifuge at 4,000 rpm for

20 min and remove the supernatant completely by inverting the tubes. Remove

the supernatant sticking on the rim of the tube with the help of filter paper.


Estimation of Phenols


2. Dissolve precipitate in 0.4 ml of glacial acetic acid.

3. Prepare a reagent blank by taking 0.2 ml of acetic acid and 5 ml of reagent 1.

4. Run a standard by taking 0.1 ml acetic acid, 0.1 ml reagent 5 and 5 ml of reagent

1. Take 0.2 ml of the dissolved precipitate and add 5 ml of reagent 1. Mix the

contents thoroughly and allow it to stand at room temperature for 10 min. and


5. Take 2 ml of reagent 2 in another set of tubes; add 3 ml of the supernatant

obtained from above. Mix the contents thoroughly; cool the tubes to room


6. Read the absorbance at 560 nm against a reagent blank.

Normal Values

Total cholesterol – 150–250 mg/dl

HDL cholesterol – 30–70 mg/dl

LDL + VLDL Cholesterol – 90–210 mg/dl


(VLDL + LDL) cholesterol in serum (mg/100 ml)


Absorbance of test sample


Absorbance of standard

Clinical Interpretations

HDL cholesterol is regarded as a risk factor for coronary heart diseases (CHD). The

level of HDL cholesterol is found to be decreased in patients suffering from

coronary heart disease, atherosclerosis, diabetes mellitus and in smokers also.

It is reported that HDL cholesterol increases during physical exercise, alcohol

intake, oestrogen therapy and also insulin therapy in diabetics.

Women are found to have slightly higher values of HDL cholesterol.

Ratio of HDL cholesterol to total cholesterol decreases as in CHDs, diabetes

mellitus, smokers, atherosclerosis and this ratio increases in physically active persons.


Estimation of Phenols

Phenols, the aromatic compounds with hydroxyl groups are widespread in

plant kingdom. They occur in all parts of the plants. Phenols are said to offer

resistance to diseases and pests in plants. Grains containing high amount of

polyphenols are resistant to diseases. Phenols include an array of compounds

like tannins, flavonols etc.


Phenols react with phosphomolybdic acid in Folin-Ciocalteau reagent in alkaline

medium and produce blue coloured complex.



Methods for Nutritional Quality Evaluation of Food Materials






80% Ethanol

Folin-Ciocalteau reagent


Standard: 100 mg catechol in 100 ml water, dilute 10 times for a working standard.


1. Weigh exactly 0.5–1.0 g of the sample and grind it with pestle and mortar in

10-time volume of 80% ethanol.

2. Centrifuge the homogenate at 10,000 rpm for 20 min. Keep the supernatant and

re-extract the residue with 5 times the volume of 80% ethanol, centrifuge

and pool the supernatants.

3. Evaporate the supernatant to dryness.

4. Dissolve the residue in a known volume of distilled water (5 ml).

5. Pipette out different aliquots (0.2–2 ml) into test tubes.

6. Make up the volume in each tube to 3 ml with water.

7. Add 0.5 ml of Folin-Ciocalteau reagent.

8. After 3 min, add 2 ml of 20% Na2CO3 solution to each tube.

9. Mix thoroughly and place the tubes in boiling water for exactly 1 min, cool and

measure the absorbance at 650 nm against a reagent blank.

10. Prepare a standard curve using different concentrations of catechol.


From the standard curve find out the concentration of phenols in the test sample and

express as mg phenols/100 g material.


1. If any white precipitate is observed on boiling, the colour may be developed at

room temperature for 60 min.

2. Express the results in terms of catechol or any other phenol equivalents used as



Chlorogenic Acid (Michael et al. 1978)

Phenolic compounds are widely distributed in plants. They contribute to the colour

and flavour of plant parts. The presence of phenolic compounds in oilseeds and

grains pose nutritional problems.


Chlorogenic acid is extracted with alcohol, dried and dissolved in acetone. It is

reacted with titanium ion to form a coloured complex which is measured at 450 nm.


Estimation of Tannins








Titanium Reagent: 20% TiCl4 in con. HCl

Standard 25–200 mg/ml Chlorogenic acid in Acetone


80% Ethanol

2.5 N HCl


1. Reflux twice a known quantity of defatted sunflower meal in 80% ethanol

(adjusted to pH 4.0 with 2.5 N HCl) for 30 min (125 ml to 1 g meal).

2. Discard the precipitate and collect 250 ml of the extract.

3. Remove 0.5 ml samples and dry in a vacuum oven at 50 C and 700 mm pressure

for 2 h.

4. Dissolve dried extract in 4.7 ml of acetone.

5. Add 0.25 ml of TiCl4.

6. Read the colour at 450 nm against a reagent blank (acetone plus TiCl4).

7. Similarly treat the standards with TiCl4 and read the colour intensity.

8. Draw a standard curve and find out the chlorogenic acid content in the sample.


Express chlorogenic acid content as grams per 100 g sample.


Estimation of Tannins

Tannins and tannin-like substances are widespread in nature and are probably

present in all plants. These are polyphenolic compounds and are divided into two

main groups-hydrolysable and condensed.

1. Hydrolysable tannins contain a polyhydric alcohol usually, if not always,

glucose esterified with gallic acid or with hexahydroxydiphenic acid.

2. Condensed tannins are mostly flavonols and are probably polymers of flavan

3-ol (catechin) and these cannot be hydrolyzed to simple components.

Although the seeds with high polyphenol content seeds are resistant to common

diseases, they also display anti-nutritional attributes. Among the cereals, sorghum

contain higher amounts of polyphenols

The tannins are estimated by the following two methods:

(i) Folin-Denis Method: This is based on the non-stoichiometric oxidation of the

molecules containing a phenolic hydroxyl group.

(ii) Vanillin Hydrochloride Method: Vanillin method is specific for dihydroxyphenols and is particularly sensitive for meta-substituted, di and tri

hydroxybenzene containing molecules.



Methods for Nutritional Quality Evaluation of Food Materials

(i) Folin-Denis Method


Tannin-like compounds reduce phosphotungstomolybdic acid in alkaline solution

to produce a highly coloured blue solution, the intensity of which is proportional to

the amount of tannins. The intensity is measured in a spectrophotometer at 700 nm.


1. Folin-Denis Reagent: Dissolve 100 g sodium tungstate and 20 g phosphomolybdic

acid in 750 ml distilled water in a suitable flask and add 50 ml phosphoric acid.

Reflux the mixture for 2 h and make up to 1 l with water. Protect the reagent from

exposure to light.

2. Sodium Carbonate Solution: Dissolve 350 g sodium carbonate in 1 l of water at

70–80 C. Filter through glasswool after allowing it to stand overnight.

3. Standard Tannic Acid Solution: Dissolve 100 mg tannic acid in 100 ml of

distilled water.

4. Working Standard Solution: Dilute 5 ml of the stock solution to 100 ml with

distilled water. One ml of solution would contain 50 mg tannic acid.


• Extraction of Tannin: Weigh 0.5 g of the powdered material and transfer to a

250 ml conical flask. Add 75 ml water. Heat the flask gently and boil for 30 min.

Centrifuge at 2,000 rpm for 20 min and collect the supernatant in 100 ml

volumetric flask and make up the volume.

• Transfer 1 ml of the sample extract to a 100-ml volumetric flask containing

75 ml water.

• Add 5 ml of Folin-Denis reagent, 10 ml of sodium carbonate solution and dilute

to 100 ml with water. Shake well and read the absorbance at 700 nm after

30 min.

• Prepare a blank with water instead of the sample.

• If absorbance is greater than 0.7, make appropriate dilution.

• Prepare a standard graph by using 0–100 mg tannic acid.


Calculate the tannin content of the samples as tannic acid equivalents from the

standard graph.

(ii) Vanillin Hydrochloride Method


The vanillin reagent will react with any phenol that has an unsubstituted resorcinol

or phloroglucinol nucleus and forms a coloured substituted product which is

measured at 500 nm.


Estimation of Lignin



1. Vanillin Hydrochloride Reagent: Mix equal volumes of 8% hydrochloric acid in

methanol and 4% vanillin in methanol. The solutions must be mixed just before

use, and avoid using even if it is slightly coloured.

2. Catechin-Stock Standard Solution: Prepare a standard solution containing 1 mg

catechin/ml methanol.

3. Working Standard: Dilute the stock solution 10 times from 10 to 100 ml

(100 mg/mL).

4. Preparation of the Extract: Extract 1 g of ground seed in 50 ml methanol. Mix

occasionally by swirling. After 20–28 h, centrifuge and collect the supernatant.


1. Pipette out 1 ml of the supernatant and quickly add 5 ml of vanillin hydrochloride reagent. Take readings in a spectrophotometer at 500 nm after 20 min.

2. Prepare a blank with vanillin hydrochloride reagent alone.

3. Prepare a standard graph with 20–100 mg catechin using the diluted stock



From the standard graph, calculate the amount of catechin, i.e. tannin in the sample

as per the absorbance values and express the results as catechin equivalents.


Estimation of Lignin (Goering and Van Soest 1975)

Lignins constitute around 30% of the organic matter of trees and besides other

functions acts as a physical barrier against invading pathogens. It is present in the

cell walls of plants which along with cellulose contribute rigidity and stiffness to

plant stems.


Refluxing the sample material with acid detergent solution removes the watersoluble and materials other than the fibrous component. The left-out material is

weighed after filtration, dried, treated with 72% H2SO4 and filtered, dried and

ashed. The loss of weight on ignition gives the acid detergent lignin.


1. Acid detergent solution: Dissolve 20 g of cetyl trimethyl ammonium bromide

(CTAB) in 1 l of 1 N sulphuric acid.

2. Sintered glass crucible-G2

3. 72% H2SO4 (w/v)







Methods for Nutritional Quality Evaluation of Food Materials


Round bottom flask

Refluxing Set

Muffle Furnace


C. Acid Detergent Fibre (ADF)

1. Place 1 g of powdered sample in a round bottom flask and 100 ml of acid

detergent solution. Heat to boil in 5–10 min. Reduce heat to avoid foaming as

boiling begins. Reflux for 1 h after the onset of boiling. Adjust boiling to slow,

even level.

2. Remove container, swirl and filter the contents through a pre-weighed sintered

glass crucible (G-2) by suction and wash with hot water twice.

3. Wash with acetone breaks up the lumps. Repeat acetone washing until the filtrate

is colourless.

4. Dry at 100 C for overnight.

5. Weigh after cooling in a desiccator.

6. Express ADF content in percentage i.e. W/S x 100, where W is the weight of the

fibre and S is the weight of the sample.

D. Determination of Acid Detergent Lignin (ADL)

1. Transfer ADF to a 100-ml beaker with 25–50 ml of 72% sulphuric acid. Add 1 g

asbestos. Allow it to stand for 3 h with intermittent stirring with a glass rod.

2. Dilute the acid with distilled water and filter with pre-weighed Whatman No. 1

filter paper. Wash the glass rod and the residue several times to get rid of the


3. Dry the filter paper at 100 C and weigh after cooling in a desiccator.

4. Transfer the filter paper to a pre-weighed silica crucible and ash the filter paper

with the content in a muffle furnace at 550 C for about 3 h.

5. Cool the crucible in a desiccator and weigh. Calculate the ash content.

6. For blank, take 1 g asbestos, add 72% H2SO4 and follow the steps 2–5.

• For preparing pre-weighed filter paper, see the note under pectic substance.

NDF ẳ Hemicellulose + Cellulose + Lignin + Minerals

ADF ¼ Cellulose + Lignin + Minerals

Hemicellulose ¼ NDF – ADF

Cellulose ¼ ADF – Residue after extraction with 72% H2SO4

Lignin ¼ Residue after extraction with 72% H2SO4 – ash

• Wet materials can also be used for lignin estimation, provided the wet sample

is equivalent to 1 g of dry material.

• If the weight loss of asbestos blank on ashing is below 0.002 g/g, discontinue

the determination of blank.

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26 Estimation of High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) and Very Low Density Lipoprotein (VLDL) Cholesterol in Serum

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