Biochemistry for Medical Laboratory Science

MCMT 20/200
Harold John A. Borlaos
Chapter 1: “Carbohydrates”
A. General Characteristics of Carbohydrates:

 Contains Carbon, Hydrogen and Oxygen

 Contains C=O and -OH
 Empirical Formula: Cx(H2O)y
 Most abundant organic molecules in nature
 Glyceraldehyde is the smallest carbohydrates

B. Functions:

 Major Energy Source: Glucose

 Storage form of glucose: Glycogen
 Components of cell membranes: Glycoproteins
 Structural component in plants, bacteria and insects: Chitin and cellulose

C. Classification of Carbohydrates

C1: According to the number of sugar units

CLASS STRUCTURE EXAMPLE

Monosaccharides Contains 1 sugar unit Glucose
Galactose
Fructose
Cannot be broken down into simpler units by
hydrolysis reactions

Disaccharides 2 sugar units linked together by a glycosidic Maltose (Glucose + Glucose)

Lactose (Glucose + Galactose)
bond
Sucrose (Glucose + Fructose)
Contains two monosaccharides

Oligosaccharides Contains 3-10 monosaccharides or sugar Seldom encountered

units

Polysaccharides More than 10 sugar units Glycogen

Starch
Chitin

C2: According on Reducing Property

Reducing sugar Glucose, Maltose, Lactose, Fructose, Galactose

Non-Reducing sugar Sucrose

 C3: According to Functional Group

Aldose Ketose
Contain and Aldehyde group Contain a Ketone group
Glucose, Galactose, Ribose Fructose

C4: According to Stereochemistry of the compound

Stereochemistry Description Example

1. Isomers  Compounds that have the same Glucose, fructose, galactose and
chemical formula mannose are all isomers of one
another because they have the same
formula: C6H12O6
2. Epimers  Isomers that differ in Glucose and Galactose (differ only in
configuration around only one position of -OH in C4)
specific carbon atom (except Glucose and Mannose (differ only in
the carbonyl carbon) position of -OH in C2)
3. Enantiomers  Optical isomers or
stereoisomers
 Pair of structures that are
mirror images of each other
 Designated as a D-sugar
(Dextrorotatory) and L- sugar
(Levorotatory)
 D-sugars are more common

4. Anomers  In aqueous solution, A-D-glucopyranose

monosaccharides with five or B-D-glucopyranose
more carbon atoms occur
predominantly as cyclic/ ring
structure.
 Furanose: monosaccharide
structure with five-membered
 ring
 Pyranose: monosaccharide
structure with six-membered
ring
 Rotation around the carbonyl
carbon produces anomers
which are labeled as alpha
anomers (-OH in carbonyl
carbon is in downward
position) and beta anomers (-
OH in carbonyl carbon is in
upward position)

Epimers at C2 of D-glucose: D-Mannose

Epimers at C3 of D-glucose: D-Allose

Epimers at C4 of D-glucose: D-Galactose

Epimers at C5 of D-glucose: L-Idose

ANOMERS
Activity 1: Classify each carbohydrate as a Monosaccharide, Disaccharide or Polysaccharide

Activity 2: Classify each of the following monosaccharides according to both the number of carbon
atoms and the type of carbonyl group present
D. Carbohydrate Metabolism

 Amylase catalyze the hydrolysis of starch to maltose (a molecule composed of two glucose)
 Salivary amylase (Ptyalin) and Pancreatic amylase (Amylopsin)
 Disaccharides are further hydrolyzed into monosaccharides by specific enzymes

Disaccharides Other Name Enzymes Products

Sucrose Table Sugar Sucrase (Glucose + Fructose)
Lactose Milk Sugar Lactase (Glucose + Galactose)
Maltose Malt Sugar Maltase (Glucose + Glucose)

 Glucose is the only carbohydrate to be used directly for energy

 After glucose enters the cell, it undergoes phosphorylation into glucose-6-phosphate through
the action of Hexokinase

 Glucose-6-phosphate is then shunted into the following metabolic pathways:

a. Glycolysis (Embden-Meyerhof pathway)
b. Glycogenesis
c. Hexose-Monophosphate shunt
Embden-Meyerhof Pathway: Glycolysis (Anaerobic)
E. Biochemical Process in Carbohydrates

Process Description
Glycolysis Metabolism of glucose molecule to pyruvate or lactate for production of energy
Gluconeogenesis Formation of glucose-6-phosphate from non-carbohydrate source
Glycogenesis Conversion of glucose to glycogen for storage
Glycogenolysis Breakdown of glycogen to glucose for use as energy
Lipogenesis Conversion of carbohydrates to fatty acids
Lipolysis Decomposition of fats

F. Regulation of Carbohydrate Metabolism

1. Insulin

 Produced by the beta cells of the islets of Langerhans (pancreas)

 Lowers blood glucose in the body
 The only Hypoglycemic agent
 Target: most cells of the body

Goal: Decrease blood glucose

Actions:

 Increase utilization of glucose by the cells by increasing cellular uptake

 Increase glycogenesis and inhibits glycogenolysis
 Inhibits gluconeogenesis
 Stimulates lipogenesis while inhibiting lipolysis

2. Glucagon

 Produced by the alpha cells of the islets of Langerhans

 Increases blood glucose in the body
 Target: Liver

Goal: Increase blood glucose

Actions:

 Promotes liver glycogenolysis

 Increases gluconeogenesis
 Inhibits glycolysis
3. Other hormones involved

Hormones Actions
Cortisol (Glucocorticoids) Increases gluconeogenesis
Decrease Glucose uptake and utilization by
extrahepatic tissues
Catecholamines Stimulates Glycogenolysis
Thyroid hormones Increase glucose absorption in small intestines
Growth hormone Increase liver gluconeogenesis
Inhibits glycolysis
Somatostatin Produced by the delta cells of the islets of
Langerhans (pancreas)
Inhibits glucagon and insulin secretion

G. Organ Regulation of Glucose

a. Liver: regulates and maintains the constancy of the circulating

glucose levels at around 70-110mg/dL

b. Kidneys: has a renal threshold for glucose at 170-180 mg/dL

H. Diabetes Mellitus

 Elevated fasting blood glucose caused by a relative or absolute deficiency of glucose

Sign and Symptoms

Polyuria: increased urine output

Polydypsia: excessive thirst as a compensation for polyuria

Polyphagia: “Hunger in the midst of plenty” excessive appetite because of the failure of
glucose to enter the peripheral tissues

Glucosuria: Glucose concentration exceed the renal threshold for glucose (180mg/dL)

Unexplained weight loss

Criteria for the Diagnosis of Diabetes Mellitus

Parameter Diagnostic Value

Fasting Blood Glucose >126 mg/dL
Random Blood Glucose > 200 mg/dL+ primary symptoms of DM
2-hour Plasma Glucose > 200 mg/dL during the OGTT
HbA1c >6.5%
 Type I DM Type II DM

Description Characterized by an absolute deficiency Characterized by a combination of

of insulin caused by an autoimmune insulin resistance and dysfunctional
attack on the beta cells of the pancreas beta cells

Also Known as Juvenile-Onset DM Adult-Onset DM

Insulin Dependent DM Non-insulin dependent DM

Frequency 5-10% 90-95% (Most common type)

Age of Onset Common in children and young adults Common with advancing age

Risk Factors Autoimmune, Genetic, Environmental Genetic, Obesity, Sedentary lifestyle,

race/ethnicity

C-peptide level Very low or undetectable Detectable

Built Thin individuals Obese individuals

Ketoacidosis Prone to ketoacidosis and diabetic Not prone to ketoacidosis

compilations

Medication or Therapy Insulin absolutely necessary Oral agents

Multiple daily injections or insulin pump

Pathogenesis Destruction of pancreatic beta cells, No autoimmunity

usually autoimmune Insulin resistance and progressive
insulin deficiency
1. Anti-islet cell cytoplasmic
autoantibodies 1. Insulin resistance syndrome
2. Insulin autoantibodies 2. Pancreatic B cell dysfunction
3. Anti-GAD (glutamic acid
decarboxylase)

Types of Diabetes Mellitus

 “Other Specific types of Diabetes Mellitus”

A. Cushing Syndrome (Hyperadrenalism)

 A disease affecting the adrenal cortex causing excessive secretion of diabetogenic

glucocorticoids.

B. Pheochromocytoma:

 A tumor affecting the chromocytes of adrenal medulla. Adrenalin and Noradrenalin are
produced in excessive amounts which tend to cause hyperglycemia.

C. Acromegaly:

 A disease characterized by excessive enlargement of the bones of the hands, feet skull
due to overproduction of Growth hormones which elevates blood sugar.

“Gestational Diabetes”
Gestational Diabetes

 Glucose intolerance with onset or first recognition during pregnancy

 Large percent of patients develop Diabetes Mellitus within 5-10 years
 Due to metabolic and hormonal change
 Screening test: 2-hour OGTT using a 75g glucose load

Fasting Plasma Glucose 2-Hr plasma glucose level (after 75g Hba1c
of load)
mg/dL mmol/L mg/dL mmol/L %
Normal <100 <5.6 <140 <7.8
Pre-diabetes 5.7-6.4
 IFG 100-125 5.6-6.9
 IGT 140-199 7.8-11.0
Diabetes >126 >7.0 >200 >11.1 >6.5
Mellitus
 Guidelines for Oral Glucose Tolerance Test
 Carbohydrate consumption should be at least 150g/ day during the 3 -day preparation.
 No limitation in physical activity.
 Test should be performed after an overnight 8-14 hours.
 The individual should not eat food, drink tea, coffee alcohol, or smoke cigarettes during the test.
 Venous glucose samples are preferably collected in gray-top tubes containing fluoride and an
anticoagulant.
 Fasting Blood Glucose is measured right before the administration of the glucose load. A Fasting
Blood Glucose of greater than 140 mg/dL, should stop the test immediately. Proceed with the
glucose load if Fasting Blood Glucose is less than 140 mg/dL.
a. Glucose load for adults: _______________
b. Glucose load for children: ________
c. Pregnant women: ______________
 Patient should finish glucose load within 5-15 minutes.
 Patient should NOT vomit. If patient vomits, ____________________

“Glycosylated Hemoglobin”
Glycosylated Hemoglobin (HbA1c)

 Monitors glucose over the previous 3 months

 For every 1% increase in HbA1c, there is a corresponding 35mg/dL change in plasma glucose
 HbA1c should be tested at least twice a year to monitor long term glycemic control
 Specimen: EDTA: Whole blood
 False decreased: decreased RBC lifespan

“Fructosamine”
Fructosamine (Glycated Albumin)

 Monitor glucose control over past 2-3 weeks

 Albumin has a life span of 20 days in circulation
 False decreased in patients with Hypoalbuminemia

Hypoglycemia:

 Blood glucose is below 50-55mg/dL

 Warning signs: Increased hunger, sweating,
Nausea and vomiting, Dizziness, nervousness
Mental confusion and blurring of sight and speech
 Specimen Consideration and Patient Preparation
 Whole blood, serum, plasma, urine, CSF, serous fluid, synovial fluid can be used as sample
 Standard Clinical Specimen: Venous plasma glucose
 Fasting blood sugar should be obtained after 8-10 hours of fasting
 Venous blood has a lower glucose level compared to arterial blood
 Capillary Blood has higher glucose level compared to venous blood
 Whole Blood gives approximately 10-15% lower glucose levels than serum or plasma
 Glucose is metabolized at room temperature at a rate of 7 mg/dL/hour
 At 4degree Celsius, glucose decreased by approximately 2 mg/dL/ hour
 Gray top tubes are preferred for plasma glucose collection.
a. Sodium fluoride: serve as anti-glycolytic agent
b. Potassium oxalate: serve as anticoagulant
 CSF glucose concentration is approximately 60-70% that of plasma concentration
 As little as 10% contamination with 5% dextrose will elevate glucose in a sample by 500mg/dL
or more

“Carbohydrates Methodology”
I. Enzymatic Methods
A. Hexokinase
 Reference method for glucose
 Most sensitive and high specificity with lesser interferences compared to Glucose oxidase
 Measures both alpha and beta glucose
 Phosphorylation reaction (needs ATP)
 Not affected by Uric acid or Vitamin C
 NADH is directly proportional to glucose concentration and is measured via spectrophotometer
at a wavelength of 340 nm.
B. Glucose oxidase method

 Reacts only with the B-D glucose

 Polarographic method: measure the rate of disappearance of oxygen using an oxygen electrode
 Colorimetric method: use a side reaction that consumes Hydrogen peroxide

Notes in Enzymatic method!!!

1. Glucose in the solution exists either as an alpha or beta glucose

a. Alpha glucose: 35%
b. Beta glucose: 65%
2. Glucose Oxidase is specific only to beta glucose
3. Alpha glucose is converted to beta glucose by the enzyme Mutarotase
4. Source of Glucose-6-phosphate dehydrogenase: Leuconostoc mesenteroides
5. Source of Glucose dehydrogenase: Bacillus cereus

Conversion factor for glucose: mg/dL-mmol/L

0.0555
II. Chemical methods by Oxidation-Reduction
A. Based on Copper Reduction

 Glucose in hot alkaline solution readily reduces cupric ion to cuprous ions forming cuprous oxide
 Cuprous oxide is then couped with a color indicator

1. Folin Wu Method

 Reducing substances contributes major interference which it falsely increases the value of
glucose.
 Proteins are removed by tungstic acid

2. Nelson-Somogyi Method

 The most accurate Reduction-Oxidation test for glucose

 Measures TRUE glucose (Removal of non-glucose reducing substances as specimen is
deproteinized either with Barium hydroxide or Zinc sulfate.

3. Neocuproine Method

 Neocuproine reagent: 2,9-dimethyl-1,10-phenanthroline hydrochloride

4. Benedict`s Method

 Modification of Folin Wu method for qualitative urine glucose

5. Shaffer-Hartman Somogyi

 Uses the principle of Iodine reaction with cuprous byproduct and excess I2 is then titrated with
Thiosulfate

B. Based on Ferric Reduction

 Glucose in hot alkaline solution reduces yellow ferricyanide to form a colorless ferrocyanide

1. Hagedorn-Jensen Method

 First method adopted by Technicon Auto Analyzer

 Measurement is based on the decreased coloration of ferricyanide
 Reducing substances particularly uric acid and creatinine will falsely increase the result
C. Based on Condensation

1. Dubowski or O-toluidine method (Condensation of amines)

 Glucose is allowed to react with ortho-toluidine in hot glacial acetic acid to produce a bluish-
green colored N-glycosylamine plus a Schiff`s base.
 Galactose, mannose aldohexose reacts with O-toluidine which can interfere the result

2. Anthrone`s test (Condensation of phenols)

 Glucose is converted into hydroxymethylfurfural in hot strongly acidic solution. The aldehyde
group reacts with the enol tautomer of anthrone to form a bluish- green product
 “Lipid Chemistry”
General characteristics:

 Also known as Fats

 Soluble in non-polar organic solvents such as ether and chloroform
 Insoluble in polar solvents such as water
 Composed of mostly Carbon-hydrogen bond
 Water-insoluble and is transported by lipoproteins
Functions:

 Rich source of energy: Gluconeogenesis

 Integral part of the cellular membrane: phospholipids
 Precursor to steroid hormones: cholesterol
 Insulators to allow nerve conduction or to prevent heat loss

“Forms of Lipids”
A. Fatty acids
 Building blocks of lipids
 Has carboxyl group (polar/ hydrophilic) and a hydrocarbon chair (non-polar or
hydrophobic)
 Only few exists as free/ unbound fatty acids, most are bound to albumin
 Some are found as part of triglycerides and phospholipids
Saturated Fatty acids:
 If there are only single bonds
in the chain.
Unsaturated Fatty acids:
 If there are carbon-carbon
double bonds in the chain
 Types of Fatty acid
Saturated Fatty Acids
1. Saturated Fatty acid
 High melting point
 Sources: animal oil  Increases both bad and good cholesterol
 Solid in form in room temperature
2. Unsaturated Fatty acid (Cis)

 Lowers bad cholesterol and increases good

Unsaturated Fatty acids cholesterol

 Low melting point 3. Trans-Fat

 Sources: plant oils
 Increases bad cholesterol and lower good cholesterol
 Liquid in form in room temperature
 Cis-are most common configuration
 Trans-fats are synthetically produced fatty acid through the process of Hydrogenation
 The notation used for fatty acids indicates the number of carbon atoms and the number of
double bonds.
 18:1 means 18-carbon fatty acids with one double bond
B. Triglycerides

 Also known as Neutral Fat

 Composition: 3 molecules of fatty acids and a molecule of glycerol
 Transmitted in the bloodstream by chylomicrons and very low-density lipoproteins (VLDL)
 Main storage form of lipid in man (adipose tissue)
 Functions: when triglycerides are metabolized, their fatty acids are released to the cells and
converted to energy and provides excellent insulation

I. Clinical significance of Triglycerides

 Measurement of TAG is used to monitor the risk for heart disease, stroke etc.
 Used to help monitor heart conditions and treatments to lower the risk of heart disease
 It is usually done as part of group test called lipid profile.

II. Methodology for Triglycerides Measurements:

A. Enzymatic Method
1. Trinder`s Reaction (Peroxidase Reaction)

 Absorbance of the Red quinoneimine dye can be measured spectrophotometrically at 500-505 nm.
 Hemoglobin (False increased) and ascorbic acid (false decreased) can also interfere with the peroxidase
step
 Bilirubin interference can be minimized by adding potassium ferrocyanide to the reaction mixture with
peroxidase. Potassium ferrocyanide reduces bilirubin into an inactive compound.

2. Winartasaputra method (Glycerophosphate dehydrogenase method)

 Absorbance of NADH can be measured at 340 nm, after the glycerophosphate dehydrogenase reaction
 Absorbance of formazan dye can be measured between 500-600 nm

3. Buculo Method (Pyruvate kinase method)

 NAD+ can be measured at 340 nm spectrophotometrically

B. Chemical Method

1. Modified Van Handel Zilversmith

 CDC: Original reference method for Triglyceride determination

Steps Reagent Purpose

Step 1a. Extraction Folch`s reagent:
(Chloroform: Ethanol mixture)
Step 1b. Adsorption Salicic acid, zeolite
Step 2: Saponification Alcoholic Potassium hydroxide or
or Hydrolysis sodium methoxide
Pretreatment of the sample to remove
the lipids from proteins
To remove non-triglyceride glycerol such
as phospholipids, monoglycerides, and
diglycerides as well as interfering
substances such as glucose and bilirubin
To cleave triglycerides molecules into
fatty acids and glycerol

Step 3: Oxidation Sodium periodate (oxidizing agent) To convert glycerol to a measurable

compound

Step 4: Colorimetry Sulfuric acid+ chromotropic acid Aids in the measurement of the
compound that forms after oxidation;
Positive result: pink chromophore measured by it`s absorbance after
measured at 500-600 nm addition of color reagent.

C. Fluorometric method

1. Hantzsch Condensation

 Measures triglycerides
 Produces yellow compound (Diacetyl Lutidine compound)
C. Cholesterol

 Not a source of energy

 Contains 4-rings and single C-H chain tail similar to fatty acids
 Precursors of 5 major classes of steroid hormones: Glucocorticoid, Androgens,
Mineralocorticoid, Estrogens, Progestins
 Found on the surface of lipid layers, synthesized in the liver

I. Forms of Lipids

a. Cholesterol esters (70%)

 Composed of cholesterol ring and fatty acids

 It undergoes esterification by LCAT (Lecithin Cholesterol Acyl Transferase)
LCAT: catalyzes the esterification of cholesterol by promoting the transfer of fatty acids from
Lecithin to cholesterol which result in the formation of lysolecithin and cholesterol ester.

b. Free cholesterol (30%)

 Unesterified cholesterol; found on the surface of lipoproteins

 Hydrophobic
 II. Methodology for Cholesterol Determination

Chemical Methods Procedure End Product Positive result

1. Liebermann- 2 mg of dry extract was Green Cholestadienyl

Burchard`s Test dissolved in acetic acid, Monosulfonic acid
heated to boiling, cooled
and then 1mL of Stabilizer: Sodium sulfate
concentrated sulfuric acid
was added along the sides of
the test tube

2. Salkowski Test 2 mg of dry extract was Red Cholestadienyl

shaken with chloroform, to Disulfonic acid
the chloroform layer sulfuric
acid was added slowly by Stablizer: Ferric chloride
the sides of the test tube

A. Chemical Methods

1-STEP 2-STEPS 3-STEPS 4-STEPS

Color development Extraction Extraction Extraction
Color development Saponification Saponification
Color development Purification
Color development

Subjected to protein Removed Removed Removed

interference

Subjected to chromogen Subjected to chromogen Partially Removed Removed

interference interference

Color difference of free and Color difference of free and Removed Removed
esterified cholesterol esterified cholesterol

Zlatkis, Zak and Boyle Carr and Drekter Abell Levy and Brodie Schoenheimer- Sperry
Rapid and can be Good except for Icteric Standard Method Reference Method
automated Serum
Procedures for Preliminary Treatments

Steps Reagents Purpose

1. Extraction Bloor`s Reagent: Cholesterol is separated from protein
Ethanol: Ether (3:1)
2. Saponification Alcoholic Potassium Hydrolyzes esters, so all cholesterol is measured as
Hydroxide free type only
3. Purification Digitonin Removes error of non-specific chromogen
interference, to precipitate free cholesterol
4. Colorimetry Color reagent Color development, measured
spectrophotometrically

B. Enzymatic Method

 Absorbance of quinoneimine dye is measured at 500 nm

 4-aminoantipyrine is also known as 4-aminophenazone

4. Phospholipids

 Similar to triglycerides except that the third position on the glycerol backbone contains a
phospholipid head group
 Contains polar and non-polar end
 Constituent of cell membranes
 Most abundant lipid in the body
 Serves as a surfactant

Forms:

a. Lecithin or phosphatidyl choline (70%)

b. Sphingomyelin (20%)

c. Cephalin (10%)
Sphingomyelin

 Only phospholipid in membrane that is not derived from glycerol but from an amino acid alcohol
called sphingosine
 It accumulates in the liver and spleen of patients suffering from Niemann-pick disease

“LIPID STORAGE DISEASE”

Characterized by accumulation of lipid in cytoplasm of monocyte, macrophage and histiocyte

1. Gaucher`s disease: (Wrinkled or Crumpled Cytoplasm)

 Deficiency in Glucocerebrosidase or Beta-glucosidase
 Accumulation of glucocerebroside

2. Niemann-Pick disease: (Foamy Cytoplasm)

 Deficiency in Sphingomyelinase
 Accumulation of sphingomyelin

3. Tay-Sachs disease: (Vacuolated cytoplasm)

 Deficiency in Hexosaminidase A
 Accumulation of glycolipid and ganglioside

4. Sandhoff disease: (Vacuolated cytoplasm)

 Deficiency in Hexosaminidase A and B

 Accumulation of glycolipid and ganglioside

5. Sea Blue Histiocytosis

 Unknown enzyme deficiency

 Lipoproteins
“The Lipid transporter”

Lipoproteins

 Proteins that act to transport lipids

 Main purpose is to transport Triglycerides and Cholesterol to sites of energy storage and
utilization.

MAJOR LIPOPROTEINS
Chylomicrons  Largest and least dense
 Transports Exogenous triglycerides
 Apolipoprotein: ApoB-48
 Produced in the intestine from dietary fats

Methods:
 Standing Plasma Test/ Overnight Standing Test/ Refrigerator Test
 Done if the sample is turbid
 Stored at 4 degrees Celsius
 Positive result: Floating Creamy Layer
VLDL  Also known as Pre-Beta Lipoprotein
 Secreted in the liver
 Transports Endogenous triglycerides
 Apolipoprotein: ApoB-100
 Positive result: Turbid
HDL  Smallest lipoproteins but the most dense
 Also known as the good cholesterol
 Transport excess cholesterol from the tissue and return it to the
liver (Reverse cholesterol transport)
 Apolipoprotein: ApoA-1
 Produced in the liver and intestines
 Reference method: Ultracentrifugation
LDL  Major end product from the catabolism of VLDL
 Synthesized in the liver
 Transport cholesterol from the liver to the peripheral tissues
 Primary marker for Coronary Heart Disease
 Also known as Bad cholesterol
 Apolipoprotein: Apo B-100

Intermediate Density Lipoprotein or IDL
Lipoprotein A or Lp-A
B-VLDL
Lipoprotein-X
Ultracentrifugation
Electrophoresis
LDL cholesterol
Quantitation
Minor Lipoproteins
 Product of VLDL catabolism or remnant of VLDL
 Can be seen in Type III Broad Beta Lipoprotein
disease
 Migrates either in the pre-B or B region
(electrophoresis)
 Major lipoprotein: ApoB-100
 Known as the sinking pre-B Lipoprotein
 Composition and components similar to LDL
 Increased level may indicate premature Coronary
Heart Disease and stroke
 Floating B Lipoprotein
 Abnormally migrating B-VLDL
 Found in type III Hyperlipoproteinemia or
dysbetalipoproteinemia
 Found in obstructive jaundice and LCAT deficiency
 Specific and sensitive indicator of cholestasis
Lipoprotein Methodologies
 Reference method for quantitation of lipoproteins
 Expressed in: Svedberg units
 Reagent: Potassium iodide with 1.063 density
Electrophoretic pattern of lipoproteins:
 Origin: chylomicrons
 Beta: LDL -slowest migrating lipoproteins
 Pre-beta: VLDL
 Alpha: HDL -fastest migrating lipoprotein
Preferred supporting medium: Agarose gel
LDL= Total cholesterol- HDL-VLDL
Where: VLDL=Triglycerides/5
a. Friedwald`s Formula:
 VLDL= TAG/2.175 (mmol/L)
 VLDL= TAG/ 5.0 (mg/dL)
Note: Limitation of Friedwald: Tag is >400 mg/dL
b. DeLong`s Formula:
 VLDL= TAG/2. 825 (mmol/L)
 VLDL= TAG/ 6.5 (mg/dL)
 Fredrickson-Levy Hyperlipoproteinemia
Type Frequency Disease Plasma Appearance Lipoprotein and Lipid
pattern
1 Rare Hyperchylomicronemia Creamy layer on clear Extremely elevated Tag due
Familial LPL deficiency plasma to the presence of
Chylomicrons
2a Common Familial Hypercholesterolemia Clear Elevated LDL
2b Common Familial Combined Hyperlipidemia Clear/ slightly turbid Elevated LDL and VLDL
3 Rare Familial Dysbetalipoproteinemia Clear/ slightly turbid Elevated cholesterol, Tag,
Broad Beta disease VLDL, LDL
Remnant Removal Disease Presence of B-VLDL and IDL
4 Common Familial Hypertriglyceridemia Clear or turbid Elevated Tag due to VLDL
5 Rare Mixed Hyperlipidemia Creamy layer on Elevated VLDL and increase
turbid plasma presence of chylomicrons

“Hypolipoproteinemia”

1. Tangier`s disease

 Complete absence of HDL due to a mutation in the ABCA-1 gene on chromosome 9

“The World of Proteins”

Proteins

 Polymers of amino acids joined together by ______________

 Synthesized in the liver and secreted by the hepatocyte except ________________ (secreted by
the _____________)
 Peptide bond: amide linkages formed between the carboxyl group of one amino acid and the
amino group of another.
 _______________ is the only element that distinguishes protein from other biomolecules
 Composed h Carbon, Hydrogen, Oxygen and __________
 Most abundant macromolecules in the body

Amino acids:

 The monomers or building blocks of proteins

 A. Protein Fractions

Protein Fractions
Prealbumin Prealbumin
Albumin Albumin
Alpha 1 Globulins a-1 antitrypsin
a-1 fetoprotein
a-1 lipoprotein
a-1 acid glycoprotein (____________)
Alpha 2 Globulins a-2 macroglobulin
Ceruloplasmin
Haptoglobin
Beta Globulins Transferrin
Hemopexin
B-2 macroglobulin
C-reactive protein
LDL
VLDL
Complement System
__________ (seen in plasma but not in serum)
Gamma Globulins Immunoglobulin G
Immunoglobulin A
Immunoglobulin M
Immunoglobulin D
Immunoglobulin E

B. Diagnostically Significant Serum Proteins

Protein Description
Indicator of malnutrition, binds thyroid hormones and retinol-binding
protein

Binds bilirubin, steroids, fatty acids, major contributor to oncotic pressure

a-1 antitrypsin Protease inhibitor
a-1 fetoprotein Principal fetal protein
a-1 acid glycoprotein May be related to immune response
Binds hemoglobin
Transports copper, peroxidase activity
a-2 macroglobulin Inhibits thrombin, trypsin and pepsin
Transports iron
Binds heme
Complement For immune response by promoting cell lysis
Fibrinogen Precursor of fibrin
For inflammation and opsonin
Plasma Protein
Prealbumin (Transthyretin)  Sensitive marker of _________________________ (decreased
value)
 Transporter of T4 and Retinol (Retinol binding protein)
 Used to confirm that the specimen is really ____________
 ____________: protein found on CSF

Albumin  Highest concentration in plasma

 Binds various substances in the blood
 Maintains Osmotic pressure, plasma buffers
 It is a Negative Acute Phase Reactant
 Lowest plasma Albumin can be seen in __________________
 Increased Albumin indicates: __________________
 Major protease inhibitor
 Neutralizes trypsin- like enzymes but can destroy the alveoli
leading to emphysema
 Major component of a-1 globulin band- deficiency of AAT seen
as lack of an a-1 globulin on Serum Protein Electrophoresis
Alpha-1 fetoprotein  Initially synthesized by fetal yolk sac and fetal liver
 Screening: maternal serum
 Confirmatory: _____________
 Increased in: neural tube defects like Spina bifida, presence of
twins
 Decreased in:__________________) and Trisomy 18
A-1 acid glycoprotein  Also known as _____________
 Binds on basic dye and lipophilic hormones
 Greatest affinity to progesterone
A-1 chymotrypsin  Major transporter of _____________________
 Increased in _________________
A-2 macroglobulin  Increased in ______________________
 2nd biggest protein next to Albumin
 Proteolytic enzyme inhibitor that inhibits thrombin, trypsin
and pepsin
Haptoglobin  Bind free ______________to prevent loss of __________
And its constituents like iron into the urine
 Used primarily to help detect and evaluate _______________
Ceruloplasmin  _______________ binding protein
 Decreased in ________________ and Menkes kinky-hair
syndrome
 ___________________ deposition of coppers in skin, liver and
cornea
Transferrin  Transport two molecules of iron
 Negative phase acute reactant
 Major component of the beta globulin fraction
 Increased in pregnancy and __________________
B-2 microglobulin  Component of the major Human Leukocyte Antigen (MHC I)
 Formation of fibrin clot upon thrombin activation
  Also known as Factor 1
 Most abundant factor in coagulation
Complement  A natural defense protein that helps protect human against
infection
 Lysis of foreign agents
 _________ is the most abundant form in serum
Hemopexin  Acts by binding _______ released by hemoglobin degradation
C- reactive protein  Major acute phase reactant
 Normal to have undetected CRP in normal individual
 Increased amount indicates: _____________
Gamma globulins  Immunoglobulins are synthesized by _____________
 IgG: most abundant, can cross the placenta
 IgA: found in secretions
 IgM: first antibody to appear
 IgD: present on surface of B cells
 IgE: allergic, anaphylactic and parasitic infection