This document discusses cholesterol, including its importance, metabolism, extraction, and reduction in beef suet using lecithin. It provides details on cholesterol's properties, sources, and transport in the blood. Methods for estimating total cholesterol are described, such as spectrophotometry and gas chromatography. Cholesterol content is listed for various fish, shellfish, and mollusks. The document also examines bleaching and deodorizing beef suet, purifying soybean lecithin, and using lecithin to reduce cholesterol in beef suet by forming a lecithin-cholesterol complex. Greater cholesterol removal was achieved with higher lecithin-to-suet and stirring ratios.

1. Cholesterol: Importance ,Metabolism
and Extraction Methodology
Naresh Kumar Metha
pHd-pa1-04

2. 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"

3. Properties
Molecular
formula
C27H46O
Molar mass
386.65 g/mol
Appearance
white crystalline powder
Density
1.052 g/cm
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,isopro
pyl myristate, methanol
3

4. Importance of cholesterol
• 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

5. •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.

6. •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.
2.
3.
4.
5.
Chylomicrons-largest size and lowest density
Very Low Density Lipoproteins (VLDL)
Intermediate Density Lipoproteins (IDL)
Low Density Lipoproteins (LDL)
High Density Lipoproteins (HDL)

7. Sources of Cholesterol
Diet
De novo synthesis
Cholesterol synthesized
in extrahepatic tissues
Liver cholesterol
pool
Secretion of HDL
and VLDL
Free cholesterol
In bile
Conversion to bile salts/acids

8. •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

9. 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

10. 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 twocarbon 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 mevalonate
3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate
(IPP), with the concomitant loss of CO2
4. IPP is converted to squalene
5. Squalene is converted to cholesterol.

12. The Utilization of Cholesterol
Cholesterol 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.

13. Estimation of total cholesterol
The total cholesterol present in serum and heart was estimated
according to the method of Parekh and Jung (1970) with slight
modifications.
Reagents
1. Standard cholesterol solution (stock): 1mg/ml in chloroform
2. 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.

14. 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.

15. Gas Chromatography
Reagentsa. GC Column packing
i.
Stationary phase- J X R or OV-1 or OV-101 di methylpolysiloxane or OV-17 or
OV-22 methyl phenylpolysiloxane
ii. Support-100.200 mesh Gas-chrom Q
b.Ethylene acetate- distilled in gas
c. 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 both
g.Cholesterol β-sitosterol standard mixture- 0.6 μg cholesterol and 1.5 μg βsitosterol/ μl
h. 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.

16. Apparatusa. 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 column
c. Conditioning of column- heat 12-24 h

17. 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

18. 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

19. Cholesterol content of some fishes ,shellfish and mollusk
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
S. Mathew et al. / Food Chemistry 66 (1999) 455-461

20. 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

21. Reduction of cholesterol in beef suet using lecithin
Ali Heshmatia,*, Iraj Khodadadib
Journal of Food Composition and Analysis 22 (2009) 684–688
a Department of Food Sciences, Faculty of Agriculture, Tehran
University, Tehran, Iran
b Department of Biochemistry and Nutrition, Faculty of Medicine,
Hamadan University of Medical Sciences, Hamadan, Iran

22. 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).

23. 1. Bleaching
and deodorization of beef suet
Beef 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 |3carotene 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).

24. 2. Commercial
soybean lecithin purification
To 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.

25. Cholesterol-lowering effects of lecithin on beef suet
To 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 lecithincholesterol 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.
3.

26. 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

27. Cholesterol removal effects of different ratios of lecithin-to-suet on beef suet .
a
.
Lecithin-to-suet ratio
1:5
Cholesterol removal
(%)
40.06 ±1.72A
1:10
32.60 ±1.80B
1:20
18.87±1.91C
1:30
13.83 ±1.46D
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

28. Cholesterol removal effects of different stirring rates on beef suet
Stirring rate (rpm)
200
500
1000
1250
Cholesterol removal (%)
22.47 ±1.92D
32.60 ±1.80C
37.33 ±1.65B
42.77 ±1.82A
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.

29. Cholesterol removal effects of different stirring times on beef suet
Stirring time (h)
0.5
1.5
3
6
12
Cholesterol removal (%)
23.1 ±1.91B
32.60 ±1.80A
33.93 ±2.15A
31.73 ±2.04A
32.23 ±1.51A
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.

30. Cholesterol oxidation in traditional Mexican dried and deep-fried food
products
Journal of Food Composition and Analysis 21 (2008) 489–495
Ida 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, Mexico
b Facultad de Bioanalisis, Universidad Veracruzana, Mexico
c Dipartimento Scienze degli Alimenti, Universitd di Bologna, Italy

31. 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,
5,6bepoxycholesterol, 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´aCruset 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.

32. COPs content, cholesterol content (mg/100g sun-dried shrimp) and
extent of cholesterol oxidation (%) of sun-dried shrimp samples
COPs
7a-Hydroxycholesterol
7 b-Hydroxy cholesterol
5,6 b-Epoxy cholesterol
5,6a-Epoxycholesterol
20a-Hydroxycholesterol
7-Ketocholesterol
Cholestanetriol
25-Hydroxycholesterol
Total COPs
SH1 (mean±SD)
3.40 + 0.40a
3.50 + 0.60a
1.40 + 0.23a
1.90 + 0.35a
0.25 + 0.03a
5.03 + 0.70a
0.050 +0.001a
0.16 + 0.03a
15.90+ 0.02a
SH2 (mean±SD)
1.40 + 0.10b
1.80 + 0.10b
3.10 + 0.08b
2.30 + 0.02b
0.15 +0.01b
3.80 + 0.10b
0.12 +0.01b
0.09 + 0.01b
13.06+ 0.70b
SH3 (mean±SD)
4.4 + 0.03c
5.9 + 0.02c
3.6 + 0.02c
4.00 + 0.02c
0.33 +0.01c
6.80 + 0.10c
0.23 + 0.02c
0.20 +0.01c
25.40+ 0.01c
Cholesterol
149.23 +3.20a
131.15+ 6.40b
110.0+ 5.40c
10.60
9.95
23.00
Oxidized cholesterol (%)
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).

33. Thanks for paying
yours attention