BIOCHEMISTRY LABORATORY

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II. PROCEDURES
ACTIVITY 8: QUANTITATIVE DETERMINATION OF
ASCORBIC ACID IN VARIOUS FOOODSTUFF
➢ Fresh cabbage
Lemon Pulp
I. INTRODUCTION Orange Peel
Apple
➢ 23 VITAMINS essential micronutrients Potatoes
Polyvitamins
➢ Adequate supplies of heterogenous
nutrients are critical for health, ➢ Weigh 10 grams of the test
development, and longevity sample

➢ Vitamin K – bone matrix ➢ 1 pill of polyvitamin and dissolved

in 30 mL water
➢ Vitamin C and E – important
antioxidants ➢ 3 erlenmeyer flask, 5 mL of extract,
and 1 mL of starch solutiuon
➢ Ascorbate (Vitamin C)
➢ In burette, put Iodine solution
- powerful reducing agent
- participates in the synthesis of ➢ Brown to black tinge
chondroitin sulfate
- formation of hydroxyproline of ➢ Compute for the content of
collagen ascorbic acid in the sample (in
- wound healing, biosynthesis of mg/dL)
neurotransmitters, and
immune function
VITAMIN C
- essential for gums, arteries, soft
tissues, bone, brain, nerve
function, nutrient metabolism, • Also known as ASCORBIC ACID
antioxidant defense, and free- • Water soluble vitamin
radical scavenging • First realised more than 250 years ago
• Scurvy-killed many sailors
➢ Determined by redox titration using
Iodine
SYMPTOMS OF SCURVY
➢ Also known as Ascorbic acid

➢ When Iodine is added ascorbic acid • Pale skin

oxidized to dehydroascorbic acid, while • Sunken eyes
iodine is reduced to iodide ions • Loss of teeth

➢ Forming the blue-black starch iodine ➢ Vitamin C- playa role in many vital
complex, endpoint of titration functions like metabolism and immunity

➢ 75 mg per day (120 mg per day for

pregnant and breastfeeding women)

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➢ 2,000 mg per day is the highest daily

intake tolerable upper level (UL) WATER SOLUBLE VITAMINS

➢ Vital for growth, health, repair of bones, B1 (Thiamine) INFANTS: Dyspnea,

tendons, cartilage, and skin. Eyanosis, Diarrhea

➢ Also work as a potent antioxidant to ADULTS: Beriberi,

prevent our bodies from Wernicke Korsakoff
syndrome
➢ Human body can’t produce Vitamin C. It B2 (Riboflavin) Mouth and skin
needs to be consumed daily to maintain lesions
adequate level B3 (Niacin) Pellagra
B6 (Pyridoxine) Anemia, Seborrhea
➢ The only mammals that can’t make
B12 (Cobalamin) Megaloblostic
Vitamin C in their bodies are primates,
anemia, neural
guinea pigs, fruit bats, and humans.
abnormality
B9 (Folic acid) Megaloblostic
➢ Mammals that can make their own
anemia
Vitamin C can live eight to ten times
C (Ascorbic Acid) Scurvy
beyond their age of physical maturity

➢ Smoking causes the body to excrete
more vitamin C than usual, so smokers
need to up their in take
➢ Extremely high dosage of Vitamin C is
used by doctors as a part of cancer
treatment. In these causes the Vitamin C
is given intravenously
FAT SOLUBLE VITAMINS
A Night blindness
D Rickets (children)
Osteomalacia (adult)
E Mild hemolytic
anemia (newborn)
RBC fragility
Ataxia
K Easy bruising
Hemorrhage
CARBOHYDRATES
• Hydrates the aldehyde or ketone
derivatives based on the location of co-
functional group
• FORMS:
➢ Monosaccharides
➢ Disaccharides
➢ Oligosaccharides
➢ Polysaccharides
• Glucose is only carbohydrates to be
directly used for energy or stored as
glycogen
• Brain is completely dependent on blood
glucose for energy production, -2/3 of
glucose utilization occurs in the central
nervous system
• Glucose metabolism generates pyruvic
acid, lactic acid, and acetyl coenzyme
A as intermediate products

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• The complete oxidation of glucose yields ➢ Glycogen storage diseases

carbon dioxide, water, and adenosine ➢ Galactosemia
triphosphate or ATP ➢ Essential fructosuria
➢ G6PD deficiency
➢ PK deficiency
PATHWAYS

5 DISORDES
• Glycolysis – Glucose Pyruvate/
Lactate
• Diabetes Mellitus Type 1 (Insulin
• Gluconeogenesis – Glucose - 6- dependent)
Phosphate form non-carbohydrate
source - Result of cellular mediated
autoimmune destruction of beta cell
• Glyconeogenesis – Glycogen of the pancreas
Glucose
- Individuals at greater of developing
• Glyconeogenesis – Glucose this type of diabetes have high titers
Glycogen of multiple auto antibodies, glutamic
acid decarboxylase (GAD 65) and
• Lipogenesis – CHO Fatty acid insulin auto antibodies (IAA)

• Lipolysis – Decomposition of fat (‘di ko - Complications:

natapos isulat huhu)
QUANTITATIVE TEST/ METHOD FOR GLUCOSE
• Diabetes Mellitus Type 2
• Ortho-toluidine (Dubowski Method)
• Hexokinase - (Note: For specific test daw
sabi ni ma’am)
• Glucose oxidase - (Note: Commonly
used daw sabi ni ma’am)
CLINICAL UTILIZATION OF CARBOHYDRATES
• Diagnose Diabetes mellitus
➢ Nephropathy
➢ Neuropathy
➢ Retinopathy
- Mechanism of developing type 2 DM
- The first one is a decreased ability of
insulin to act on peripheral tissues.
Usually we call it “insulin resistance”
- The second is the dysfunction of
pancreatic B-cells represented by
the inability to produce enough
insulin to overcome insulin resistance
in the peripheral tissues

• Inborn errors of carbohydrates • Hurler Syndrome

metabolism
- Mucopolysaccharidosis type IH
(MPS-IH), genetic disorder that results
in the build up of large sugar
molecules and glycosaminoglycans

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(AKA GAGS or aldehyde -(aldoses). Aldoses may react

mucopolysaccharidosis) in
lysosomes
- The underlying mechanisms is a
deficiency of alpha-L iduronidase,
an enzyme responsible for braking
down GAGS.
• Glucose - 6- Phosphate- dehydrogenase
deficiency
- Genetic abnormality that results in an
inadequate amount of glucose -6-
phosphate dehydrogenase (G6PD) in
the blood
• G6PD a “housekeeping” enzyme
- That is particularly important for the
survival of red blood cells and their
ability to respond to oxidative stress
significantly to create a faint pink to cherry
red tint if the test is continued. Ketoses
dehydrate more quickly than aldoses.
• Benedict’s test- The sugar is changed into
enediols, a potent reducing agent, by the
presence of alkaline sodium carbonate.
Due to the creation of cuprous oxide
Cu2o, the mixture's color changes from
blue to brick-red precipitate when the
reduction reaction takes place
• Barfoed’s test- The reduction of cupric
acetate through the reduction of
monosaccharides and disaccharides is
the foundation of Barfoed's test reaction.
Brick red precipitate is produced by
reduction of cupric acetate, which also
yields cuprous oxide. Monosaccharides
often react in 1 to 5 minutes, resulting in a
crimson precipitate.

• Essential Fructosuria • Tollen’s test- The complex then oxidizes the

- Recessive disorders
fructokinase deficiency
TEST PRINCIPLES
aldehyde group of as given molecule to
results from produce a carboxylic acid. As silver ions
are converted into metallic silver, a silver
mirror appears on the bottom of a test
tube.

• Molisch test- The principle for Molisch's test

is the dehydration of sulfuric acid into
furfural. When sulphuric acid and strong
hydrochloric acid are applied to a sample
containing carbohydrate molecules, one
hydroxyl group is removed from a sugar
molecule.

• Bial’s orcinol test- This test is based on the

principle that pentoses dehydrate into
furfural, which then condenses with orcinol
to produce a bluish or blue-green
precipitate,

• Seliwanoff’s test- The Seliwanoff test is used

to distinguish between sugars with an

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ACTIVITY 10: ISOLATION OF GLYCOGEN
FROM THE LIVER OF REPLETE AND HUNGRY
ANIMAL
GLYCOGEN
• is a branched polymer of d-glucose that
stores energy and carbon in vertebrates
and is mainly found in the liver and
skeletal muscle.
• Liver glycogen serves as an immediate
source for maintaining blood glucose
levels, particularly between meals. The
glycogen stored in the liver gets
depleted after 12 to 18 hours of fasting.
• Muscle glycogen is primarily concerned
with the supply of hexoses that undergo
glycolysis to provide energy during
muscle contraction.
• Glycogen also stored disease that is
characterized by the deposition of a
normal or abnormal type of glycogen in
one or more tissues resulting in muscular
weakness, or even death
• isolation of glycogen is carried out in the
following manner: a homogenate of the
liver is prepared, and glycogen is
extracted with trichloroacetic acid (TCA)
• Proteins are denatured and precipitated
by TCA so they may be easily removed
from a solution by filtration. The liver of
animals is quickly cut to form thin plates
and they are immediately put into a
glass with boiling physiological solution
for 10-15 minutes to inactivate glycogen-
phosphorylase
ROSE MAE A. FERRER
MATERIALS, REAGENT AND EQUIPMENT
USED IN THE EXPERIMENT
• TOP LOADING BALANCE
• MORTAR AND PESTLE
• GRADUATED CYLINDER
• TEST TUBES AND RACK
• 5% TCA
• DISTILLED WATER
• LUGOL'S IODINE SOLUTION
PROCEDURES
1. WEIGH OUT 0.5G (USED 1.0 G INSTEAD) OF
THE REPLETE AND HUNGRY ANIMAL LIVER
(CHICKEN AND COW LIVER).
2. PLACE EACH SAMPLE IN A PORCELAIN
MORTAR, POUR 3 ML 5% TCA AND
POUND IT CAREFULLY FOR 10 MINUTES.
3. THEN, ADD 3ML OF DISTILLED WATER TO
THE HOMOGENATE, MIX AND FILTER
THROUGH A PAPER FILTER TO THE CLEAN
TEST TUBES.
4. MAKE THE QUALITATIVE REACTION FOR
THE DETECTION OF GLYCOGEN IN THE
FILTRATES
4.1. POUR 1 ML OF DISTILLED WATER INTO
THE FIRST TEST TUBE
4.2. POUR 1 ML OF THE FILTRATES
RECEIVED FROM THE LIVER OF A REPLETE
AND A HUNGRY ANIMAL IN THE SECOND
AND THIRD TEST TUBE RESPECTIVELY
4.3 ADD 1-2 DROPS OF LUGOL'S IODINE
SOLUTION TO EACH TEST TUBE AND
COMPARE THE COLOR OF THE TEST TUBE
CONTENTS WITH EACH OTHER
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INDICATION In what specific conditions and disease states

Iodine test for Amount of Enzyme do these pathways become utilized? Elaborate
starch starch activity level your answer.
remaining
• Glycogen storage disease type V (also
DARK BLUE - ALL NONE (0)
known as GSDV or McArdle disease) is an
BLACK
inherited disorder caused by an inability
BLUE MOST LOW (1)
to break down a complex sugar called
LIGHT SAME MODERATE
glycogen in muscle cells.
BROWN (2)
GOLD NONE HIGH (3) • A lack of glycogen breakdown interferes
with the function of muscle cells.
What important biochemical pathways related
to glycogen formation and synthesis are
happening in the liver? What hormones are necessary in regulating
glucose level in the blood? Give at least five
• The body stores carbohydrates in the and explain how these hormones affect
form of glycogen, a branched polymer. glucose homeostasis.

• Glycogen is mainly stored in the liver and

skeletal muscles and is the primary
• Insulin and glucagon are secreted from
source of energy while fasting or in
the pancreas, and thus are referred to as
between meals. The 2 metabolic
pancreatic endocrine hormones.
pathways of glycogen in the liver are
glycogenesis (glycogen synthesis) and
• Insulin helps control blood glucose levels
glycogenolysis (glycogen breakdown)
by signaling the liver and muscle and fat
cells to take in glucose from the blood.

• Glucagon increases your blood sugar

level and prevents it from dropping too
low, whereas insulin, another hormone,
decreases blood sugar levels.

• Decreasing- Insulin and amylin

• 5 Hormones

- Insulin
- Glucagon
- Amylin
- Growth hormone
- Incretin

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ACTIVITY 11: LIPIDS

• Lipids are composed of mostly carbon-

hydrogen (C-H) bonds, they are a rich
source of energy and are an efficient
way for the body to store excess
calories.

• Lipids are also precursors for the steroid

hormones, prostaglandins, leukotrienes,
and lipoxins.
ACROLEIN TEST
REAGENTS • The acrolein test detects the presence
of glycerol or fat. When fat is heated in
• Fresh Coconut Oil the presence of a dehydrating agent
• Rancid Coconut Oi such as potassium bisulphate (KHSO4),
• Corn Oil the glycerol part of the molecule
• Dichloromethane dehydrates to create acrolein, an
• KHSO4 Powder unsaturated aldehyde with a pungent,
• Ether unpleasant odor.
• Carbon Tetrachloride
• Chloroform
• Ethanol
• Diluted Hydrochloric Acid
• Diluted NaOH
• Acetic Anhydride
• Concentrated Sulfuric Acid

TEST

SOLUBILITY TEST
• Fats are soluble in organic solvents like
chloroform, alcohol, etc. It is insoluble
in water. So, if the given sample forms
an oily layer above the surface of the
water, then fat is present. Partially
soluble in alcohol and fully soluble in
chloroform than the presence of fat is
confirmed.
SPOT TEST
• A translucent spot test is a preliminary
test for lipids that is distinguished by a
transparent and greasy spot. Unlike
water, the lipid will not moisten the filter
paper. Because of their greasy texture
the lipids will penetrate the filter paper
and form a greasy or transparent spot.
Unlike lipids, the water mark will vanish
off the paper.

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UNSATURATION TEST FOR LIPIDS
• This test is used to determine whether
or not a carbon-carbon bond is
unsaturated.
• The formation of a yellow color in
samples means that it contains
unsaturated fatty acids.
LEIBERMANN - BURCHARD TEST FOR
CHOLESTEROL
• The Liebermann–Burchard test is used
for the detection of cholesterol. The
formation of a green or green-blue
colour after a few minutes is positive.
ROSE MAE A. FERRER
WHAT IS THE PRINCIPLE OF THE LIEBERMANN-
BURCHARD AND SALKOWSKI COLORIMETRIC
ASSAY? EXPLAIN ITS PRINCIPLE AND INCLUDE THE
RESPECTIVE REAGENTS USED, THE INDIVIDUAL
PROCEDURES, AND THE RESULTS.
Principle:
The Liebermann-Burchard test is a chemical
estimation test of cholesterol. It is also known as
the acetic anhydride test. In the presence of
concentrated acids, cholesterol undergoes a
reaction similar to that of alcohol, producing
colored substances.
Reagents:
Acetic Anhydride (1mL)
Sulfuric Acid (2 Drops)
Result:
Bluish-Green
Principle:
The cholesterol content of a solution can be
determined with the help of the Salkowski test.
The colors (distinct and clear colors) that result
from the interaction of cholesterol with
concentrated sulfuric acid are an important
part of the test that is used to detect
cholesterol.
Reagents:
Sulfuric Acid
Result:
Red Top layer
Yellow to Green Bottom layer
TO WHAT CLASSIFICATIONS DO CLINICALLY
IMPORTANT LIPIDS BELONG? DESCRIBE EACH
CLASSIFICATION AND ITS MEDICAL
SIGNIFICANCE.
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CHYLOMICRONS - Chylomicrons are the largest
of the four (diameter = 75-600nm). These are
synthesized by the absorptive cells of the
intestinal lining and are secreted by these cells
into the lymphatic system which joins the
circulation. Esterified cholesterol and
phospholipids
VERY-LOW DENSITYLIPOPROTEIN - Very-low-
density lipoprotein or VLDL is another class
synthesized by the liver that is analogous to the
chylomicrons secreted by the intestine. Its
purpose is to deliver triglycerides, cholesterol,
etc. to peripheral tissues.
LOW-DENSITY LIPOPROTEIN - Low-density
lipoprotein is derived from VLDL and HDL in the
plasma and contains a large amount of
cholesterol and cholesteryl ester. The
movement is from the liver to the tissue. It is also
considered the bad cholesterol.
HIGH-DENSITY LIPOPROTEIN - High-density
Lipoprotein is the smallest class. Its primary role is
in the removal of excess cholesterol from cells
and return it to the liver for degradation. It is also
significant if there is a high good cholesterol in
the body.
ELABORATE CHOLESTEROL SYNTHESIS,
ESTERIFICATION, AND CATABOLISM
Cholesterol synthesis
• takes place in the cytosol, and the
acetyl-CoA needed can be obtained
from several sources such as β-
oxidation of fatty acids, the oxidation of
ketogenic amino acids, such as leucine
and lysine, and the pyruvate
dehydrogenase reaction (acetyl-CoA
shuttled out of the mitochondria is in the
form of citrate, which is cleaved into
acetyl-CoA and pyruvate by citrate
lyase). The process of cholesterol
synthesis involves multi-steps.
ROSE MAE A. FERRER
Cholesterol esterification
• is a mechanism used by the body to
efficiently store and transport
cholesterol. Cholesterol are converted
to cholesteryl esters which are more
easily packed by the lipoproteins in
huge numbers resulting to high
availability functional cholesterol that
can be transported through the blood
stream.
Cholesterol catabolism
• is the breakdown of cholesterol which
the byproduct is being used by the
body for many functions. This occur
mainly in the liver which cholesterol is
converted into bile acids promoting
digestion of lipids and absorption of
lipid-soluble vitamins. Cholesterol can
also be converted to many sex
hormones such as androgen, estrogen
and progesterone. In the skin, oxidation
of cholesterol can be converted into
vitamin D3 by ultraviolet radiation of the
sun.
WHAT ARE THE ROLES OF LIPPROTEINS IN LIPID
METABOLISM? EXPLAIN THEIR CONTRIBUTION IN
METABOLISM
To get to the body cells, lipoproteins transport
triglycerides and cholesterol. the bad
cholesterol (LDL), which clog arteries, is
eliminated by good cholesterol (HDL).
triglycerides and cholesterol, which are
hydrophobic lipids, are moved around the
interstitial fluid and plasma by a process known
as lipoprotein metabolism. transporting lipids
through the circulatory system from sites of
utilization, is the primary role of this. rates of
cholesterol and fatty acid synthesis in tissue are
also influenced by lipoprotein constituents like
the triglycerides, fatty acids and cholesterol.
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WHAT IS ATHEROSCLEROSIS, AND HOW DO LIPIDS • HANDWASHING KILLS 100% OF BACTERIA

CONTRIBUTE TO ITS PATHOGENS
The development of atherosclerosis has been
shown to be strongly correlated with high serum
lipid level, parrticularly the elevated level of Low
Density Lipoprotein (LDL). Atherosclerosis is the
thickening or hardening of the arteries brought
on by a buildup of plaque in the inner lining of
an artery. it is widely acknowledge that the
increase LDL absorption by monocytes and
macrophages lead to the development of
atherosclerotic plaques
ACTIVITY 12: SOAP AND ITS PROPERTIES
SOAPS
• mostly prepared from blend of tallow
and coconut oils
MICELLE
• spherical arrangements of molecules in
aqueous solution such that their
hydrophobic parts are shielded from the
aqueous environment and their
hydrophilic parts are on the surface of
the sphere
SOAP SCUM
• When hard water and soap are mixed,
the magnesium ion reacts with soap
molecules and forms a solid material
called a precipitate, which does not
dissolve. This precipitate (soap scum)
reduces the ability of the soap to make
bubbles.

• In the preparation from tallow, the solid

fats of cattle are melted with steam and SOAP WORKS BEST IN
tallow layer that forms on top is removed

SAPONIFICATION

• The hydrolysis of an ester in aqueous

NaOH or KOH to an alcohol and the
sodium or potassium salt of a carboxylic
acid

• Latin: saponem “soap”

HOW SOAP CLEANS

• Soap owes its remarkable cleansing

properties to its ability to act as
emulsifying agent. Because the long
chain of hydrocarbon chains of natural
soaps is insoluble in water, soap
molecules tend to cluster in a way as to
minimize contact of their hydrogen
chains with surroundings water
molecules

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GOOD EMULSIFYING AGENT

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