Cholesterol: Importance ,Metabolism and Extraction Methodology

Naresh Kumar MethapHd-pa1-04

Cholesterol

•The name cholesterol originates from the Greek chole- (bile) and stereos (solid), and the chemical suffix - “ol ” for an alcohol.

• François Poulletier de la Salle first identified cholesterol in solid form in gallstones, in 1769.

• However, it was only in 1815 that chemist Eugène Chevreul  named the compound "cholesterine"

PropertiesMolecular formula

C27

H46

O

Molar mass 386.65 g/mol

Appearance white crystalline powder

Density 1.052 g/cm3

Melting point 148–150 °C

Boiling point 360 °C (decomposes)

Solubility in water

0.095 mg/L (30 °C)

Solubility acetone, benzene,chloroform, ethanol, ether, hexane,isopropyl myristate, methanol

• It is an essential component of life why????

• Cholesterol is the principal sterol synthesized by animals; however, small quantities can be synthesized in other eukaryotes such as plants and fungi•It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals

• It is an essential structural component of mammalian cell membranes and is required to establish proper membrane permeability and fluidity.• Cholesterol is an important component for the manufacture of bile acids, steroid hormones, and vitamin D

Importance of cholesterol

•It has been associated with the two leading causes of death in the world, heart attack and stroke.

•Coronary heart disease produces about 600000 deaths annually.

•If the cholesterol balance is well maintained between the biosynthesis, utilization, and transportation, its harmful deposition can be retained.

•Cholesterol and other substances such as trigylcerides are transported in the blood vessels in sphere-shaped body called lipoproteins.The lipoproteins are made up of five types according to size

1. Chylomicrons-largest size and lowest density2. Very Low Density Lipoproteins (VLDL)3. Intermediate Density Lipoproteins (IDL)4. Low Density Lipoproteins (LDL)5. High Density Lipoproteins (HDL)

Sources of Cholesterol

Diet De novo synthesisCholesterol synthesized in extrahepatic tissues

Liver cholesterolpool

Free cholesterolIn bile

Conversion to bile salts/acidsSecretion of HDLand VLDL

•The low density lipoproteins (LDL) is usually known as the "bad" cholesterol. It transports about 75% of the blood's cholesterol to the cells.

•LDL is usually harmless but does have dangerous interactions with the free radicals on the walls of the artery.

•The high density lipoprotein serves to remove cholesterol from the walls of the arteries. Thus, the higher level of HDL is usually better

Total Cholesterol

LDL HDL

Optimal - under 100 above 60

Desirable under 200 under 130 -

Boarderline 200-239 130-159 -

Abnormal over 240 over 160 below 35

Journal of American Medical Association, Vol 269, pp. 3015-23, 1993

Cholesterol Metabolism•Slightly less than half of the cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts for approximately 10%, and in the intestines approximately 15%, of the amount produced each day.

•Cholesterol synthesis occurs in the cytoplasm and microsomes (ER) from the two-carbon acetate group of acetyl-CoA.

The process of cholesterol synthesis has five major steps:1. Acetyl-CoAs are converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)2. HMG-CoA is converted to mevalonate3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate (IPP), with the concomitant loss of CO2

4. IPP is converted to squalene5. Squalene is converted to cholesterol.

The Utilization of CholesterolCholesterol is transported in the plasma predominantly as cholesteryl esters associated with lipoproteins. Dietary cholesterol is transported from the small intestine to the liver within chylomicrons.

Cholesterol synthesized by the liver, as well as any dietary cholesterol in the liver that exceeds hepatic needs, is transported in the serum within LDLs.

Reverse cholesterol transport allows peripheral cholesterol to be returned to the liver in LDLs. Ultimately, cholesterol is excreted in the bile as free cholesterol or as bile salts following conversion to bile acids in the liver.

Estimation of total cholesterolThe total cholesterol present in serum and heart was estimated according to the method of Parekh and Jung (1970) with slight modifications.Reagents1. Standard cholesterol solution (stock): 1mg/ml in chloroform2. Working standard: 1.0ml of the stock was diluted to 10ml with chloroform.3. FeCl3 stock solution: 10g FeCl3 dissolved in 100ml acetic acid.4. FeCl3 - H2SO4 reagent: 2.0 ml of FeCl3 stock solution was diluted to 200ml with conc. H2SO4.5. 33% KOH (W/V): 10g KOH was dissolved in 20ml distilled water.6. Alcoholic KOH solution: 6.0ml of 33% KOH was made up to 100ml with distilled ethanol. This solution was prepared fresh each time before use.

1 ml of the lipid sample was taken into a glass Stoppard tube and evaporated off the chloroform. Added 5ml freshly prepared alcoholic KOH solution. The tubes were shaken well and incubated in a water bath at 37oC for 55min. After cooling to room temperature, added 10 ml of petroleum ether and inverted the tubes once to mix the contents. Then added 5.0 ml of distilled water and shaken the tubes vigorously for 1 min. Took 0.5-2 ml aliquots from the supernatant (petroleum ether) into test tubes. Evaporated the petroleum ether extract under nitrogen. To each of the sample as well as standard tubes including the blank, added 3.0ml of glacial acetic acid followed by 0.1 ml distilled water. Mixed the tubes thoroughly and added 2.0 ml of FeCl3 - H2SO4 reagent through the sides of the test tubes. A brown ring was formed at the interface; tapped the bottom of the tubes well to effect mixing and a light colour appeared which changed to an immense purple colour and was measured in a Shimadzu – UV spectrophotometer at 560nm.The amount of total cholesterol was expressed as mg/dl in serum and mg/g in heart.

Gas Chromatography

Reagents-a. GC Column packing i. Stationary phase- J X R or OV-1 or OV-101 di methylpolysiloxane or OV-17 or

OV-22 methyl phenylpolysiloxaneii. Support-100.200 mesh Gas-chrom Qb.Ethylene acetate- distilled in gasc. Cholestane standard solution -0.4 μg/ μl (weigh 40 mg cholestane std. into 100 ml

volumetric flask and dilute to volume with ethyl acetate. d. Cholestane internal standard solution -0.2 μg/ μl dilute 10 ml std solutuion with

20 ml with ethyl acetate.e. Cholesterol standard solution -1.2 μg/ μl .weigh 60 mg cholesterol standard into

50 ml volumetric flask and dilute to volume with ethyl acetate.f. Cholestane-cholesterol standard mixture- 0.2 μg and 0.6 μg cholesterol / μl . mix

equal volume of bothg.Cholesterol β-sitosterol standard mixture- 0.6 μg cholesterol and 1.5 μg β-

sitosterol/ μlh. Cholesteryl acetate standard solution- 0.6 μg/ μl .weigh 30 mg cholesteryl acetate

standard into 50 ml volumetric flask and dilute to volume with ethyl acetate.

Apparatus-a. Gas chromatograph: Barber- Colman Co. Model 5000, Searle analytic

series 4740, or equivalent , with H2 Flame ionization detector and 1 mV strip chart recorder. Temperature(⁰) : column, 220-250; detector and flesh heater ; 240-270; flow rates,20-25 psi (138-172 kPa) to elute cholesterol in 8-12 min. H2 40-50 ml/min., air-300-340 ml/min.. Electrometer sensitivity 1 x 10-9 amp full scale deflection with 1 mV recorder.

Adjust electrometer sensitivity so that 1.5 μg cholesterol gives 50 % deflection . Repeat injections until constant peak heights are obtained on successive injections of identical volumes of standard mixture.

b. Preparation of columnc. Conditioning of column- heat 12-24 h

d. Performance – chromatograph 2 μl Cholesterol β-sitosterol standard mixture to determine retention times and resolution of column. Minimum 1600 theoretical plates is required for cholesterol peak.

theoretical plates=(L/B)2 X 16 where L= cm cholesterol peak from injection point B=cm triangulated base width of cholesterol peak Separation of cholesterol and campesterol peaks expressed as peak resolution should

be ≥ 2.2.Peak resolution =2D/(B+P) Where D is distance in cm between cholesterol and campesterol peak max. P= triangulated base width of campesterol peak B =triangulated base width of cholesterol peak

Determination –Pipette 1 ml cholestane internal standard solution into 3 dram vial containing extracted sterols, rotate vial to wash down sides with internal std solution and swirl to dissolve sterols. Inject 2 μl cholestane-cholesterol standard mixture. Identify cholesterol peak in sample from its retention time in std mixture. If cholesterol peak height in sample is >60 % full scale deflection, add additional 1.0 ml cholestane internal std solution to sample and chromatograph sample and std mixture as above. Measure cholestane and cholesterol peak height in mm.

Mg cholesterol / 100 g=(Hi /Hx) x (Cx/Ci) x (Sx/Si) x (Qi/Q) x 100

Hi and Hx height (mm) cholestone and cholesterol peaks respectively in mixture

Cx and Ci μg cholesterol and cholestane / μl reapectively in std mixture Sx and Si height (mm) cholestone and cholesterol peaks respectively in

sample Qi = μg cholestane / μl in sample and Q = mg sample/ μl

s. no. Species Cholesterol content (mg %)

1 flounder 64.7

2 Black pomfret 60.2

3 Milk fish 33.6

4 Wolf herring 39.4

5 Rohu 36.2

6 Oil sardine 86.5

7 Pink perch 56.4

8 Mackerel 69.7

9 Mackerel roe 462

10. Barracuda 34.6

Cholesterol content of some fishes ,shellfish and mollusk

S. Mathew et al. / Food Chemistry 66 (1999) 455-461

s. no. Species Cholesterol content (mg %)

11 Peneaus monodon 123

12 White shrimp 163

13 Kadle shrimp 120

14 Fiddler shrimp 143

15 Mud crab 54.8

16 Coral crab 56.5

17 Red spotted crab 52.4

18 Sand crab 66.8

19 Cuttle fish 162

20. Squid 198

S. Mathew et al. / (1999) Food Chemistry, 66 ,455-461

Reduction of cholesterol in beef suet using lecithinAli Heshmatia,*, Iraj Khodadadib

Journal of Food Composition and Analysis 22 (2009) 684–688a Department of Food Sciences, Faculty of Agriculture, Tehran University, Tehran, Iranb Department of Biochemistry and Nutrition, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

This study is aimed to investigate the effects of soybean lecithin in reducing cholesterol content of beef suet, a cholesterol-rich slaughterhouse by-product used worldwide for edible and inedible purposes, such as bakery shortenings, production of fatty acids and stock feeds, margarine and the manufacture of frying oils and soap (Haas, 2005).

1. Bleaching and deodorization of beef suetBeef fat samples were chopped into small pieces, ground and rendered in a jacketed kettle to obtain beef suet. To eliminate undesirable color caused by pigments such as |3-carotene and impurities, beef suet was heated to 95 °C and mixed with bleaching earth (1 g/100 g of beef suet) and bleached in rotary evaporator at 85 °C for 30 min (Verleyen et al., 2002). Mixture was then filtered using a Whatman filter paper in a vacuum oven at 60 °C. Finally, bleached beef suet was deodorized under N2 gas at 180 °C and stored at -20 °C (Greyt and Kellens, 2005).

2. Commercial soybean lecithin purificationTo eliminate impurities, commercial soybean lecithin (10 g) was warmed up at 50 °C and 40 mL of acetone was added with stirring for 5 min to precipitate lecithin and phospholipid contents. Liquid phase was then discarded and the pellet was washed with acetone another three times. The purified lecithin was finally vacuum-dried at 50 °C and stored at 4 °C.

3. Cholesterol-lowering effects of lecithin on beef suetTo investigate the cholesterol-lowering effects of lecithin on beef suet, lecithin paste was prepared by adding 10 g water to the 5 g of ground purified lecithin; this paste was stirred for 15 min at 500 rpm. The paste was then added to the different amounts of bleached and deodorized beef suet (25, 50,100, and 150 g), and the mixture stirred at 500 rpm for 1.5 h, allowing the lecithin-cholesterol complex to be formed. The mixture was filtered at 60 °C to exclude lecithin-cholesterol complex and to obtain beef suet with lesser cholesterol content (Kodali, 2001), the later, was dried in a vacuum oven at 60 °C and subjected to a gas chromatograph to determine cholesterol.

Fig. 1. Cholesterol removal effects of different processes on beef suet. Experiments were performed for 1.5 h at 1250 rpm and a lecithin-to-water ratio of 1:5. Values are means ± SD of three (n = 3) measurements

Lecithin-to-suet ratio Cholesterol removal (%)

1:5 40.06 ±1.72A

1:10 32.60 ±1.80B

1:20 18.87±1.91C

1:30 13.83 ±1.46D

Cholesterol removal effects of different ratios of lecithin-to-suet on beef sueta.

.

Values with different letters (A-D) within a column are significantly different at P<0.05.

a Experiments were performed for 1.5 h at 500 rpm and a lecithin-to-water ratio of 1:2.

b Values are the mean ± standard deviations of three (n = 3) experiments

Stirring rate (rpm) Cholesterol removal (%)200 22.47 ±1.92D500 32.60 ±1.80C1000 37.33 ±1.65B1250 42.77 ±1.82A

Cholesterol removal effects of different stirring rates on beef suet

Values with different letters (A-D) within a column are significantly different at P<0.05.

a Experiments were performed for 1.5 h, lecithin-to-suet ratio of 1:10, and lecithin-to-water ratio of 1:2.

b Values are the mean ± standard deviations of three (n = 3) experiments.

Stirring time (h) Cholesterol removal (%)0.5 23.1 ±1.91B1.5 32.60 ±1.80A3 33.93 ±2.15A6 31.73 ±2.04A12 32.23 ±1.51A

Cholesterol removal effects of different stirring times on beef suet

Values with letters (‘A’ and ‘B’) within a column are significantly different at P<0.05.

a Experiments were performed at 500 rpm, lecithin-to-suet ratio of 1:10, and lecithin-to-water ratio of 1:2.

Cholesterol oxidation in traditional Mexican dried and deep-fried food productsJournal of Food Composition and Analysis 21 (2008) 489–495Ida Soto-Rodrı´guezab, Perla J. Campillo-Velazqueza, Jorge Ortega-Martı´neza, Marı´a T. Rodrı´guez-Estradac, Giovanni Lerckerc, Hugo S. Garciaa,*a UNIDA, Institute) Tecnologico de Veracruz, Mexicob Facultad de Bioanalisis, Universidad Veracruzana, Mexicoc Dipartimento Scienze degli Alimenti, Universitd di Bologna, Italy

The study shows that some traditional Mexican foods (chicharron, machaca and sun-dried shrimps) have significant amounts of COPs (cholesterol oxidation products). (7a-hydroxycholesterol,7-ketocholes-terol, 5,6a-epoxycholesterol, 5,6b-epoxycholesterol, cholestanetriol, 7b-hydroxycholesterol, 20a-hy-droxycholesterol, and 25-hydroxycholesterol)

its oxidized forms or COPs have proven to be cytotoxic, mutagenic and carcinogenic (Schroepfer, 2000; O’Brien et al., 2000; Ryan et al., 2005). Furthermore, COPs have been identified as the primary factor that triggers the atherosclerotic lesion (Garcı´a-Cruset et al., 2002). Pie et al. (1991) found that the amount of COPs can reach 1–2% of the total cholesterol during daily cooking in beef, veal and pork.

Considering that these food products are widely consumed in Mexico, a large part of the population is thus exposed to COPs, and this fact could be associated to the incidence of atherosclerosis and other ailments. Development of more suitable processing and storage procedures is, therefore, necessary in order to reduce the amount of COPs in these Mexican dried and deep-fried food products.

COPs content, cholesterol content (mg/100g sun-dried shrimp) and extent of cholesterol oxidation (%) of sun-dried shrimp samples

COPs SH1 (mean±SD) SH2 (mean±SD) SH3 (mean±SD)

7a-Hydroxycholesterol 3.40 + 0.40a 1.40 + 0.10b 4.4 + 0.03c

7 b-Hydroxy cholesterol 3.50 + 0.60a 1.80 + 0.10b 5.9 + 0.02c

5,6 b-Epoxy cholesterol 1.40 + 0.23a 3.10 + 0.08b 3.6 + 0.02c

5,6a-Epoxycholesterol 1.90 + 0.35a 2.30 + 0.02b 4.00 + 0.02c

20a-Hydroxycholesterol 0.25 + 0.03a 0.15 +0.01b 0.33 +0.01c

7-Ketocholesterol 5.03 + 0.70a 3.80 + 0.10b 6.80 + 0.10c

Cholestanetriol 0.050 +0.001a 0.12 +0.01b 0.23 + 0.02c

25-Hydroxycholesterol 0.16 + 0.03a 0.09 + 0.01b 0.20 +0.01c

Total COPs 15.90+ 0.02a 13.06+ 0.70b 25.40+ 0.01c

Cholesterol 149.23 +3.20a 131.15+ 6.40b 110.0+ 5.40c

Oxidized cholesterol (%) 10.60 9.95 23.00

Each value corresponds to the mean of four replicates the standard deviation (SD) is reported. Means in the same row followed by different superscripts are significantly different according to analysis of variance and Tukey’s multiple mean comparison test (p .001).

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