2 types of adipose tissue- White adipose tissue and brown adipose tissue

o Represent the main sites of lipid storage and metabolism in the body
White adipose tissue/ cells
o Large
o Store lipids as a single large droplet
Triglycerides
o Wider distribution than brown adipose tissue
o Variations are dependent on the sex and age of the individual
o Provides insulation under the skin
o Forms cushioning fat pads around organs.
o Secretes a hormone called Leptin.
Increases carbohydrate and lipid metabolism in cells while inhibiting or
suppressing appetite and food intake
Adipose tissue/cells
o Highly vascularized as a result of high metabolic activity.
o Have receptors for insulin, glucocorticoids, and growth hormone.
Brown adipose tissue/cells
o Smaller than WAD
o Store lipids as multiple small droplets
o Found in all mammals
o Best observed in animals that hibernate
o Main function: supply the body with heat
Regulated by the sympathetic nervous system, which releases norepinephrine
to promote hydrolysis of lipids
o Gradually decreases in older individuals
o Mainly found: adrenal glands, great vessels, and in the neck region.
 Dense Irregular Connective Tissue
o Consists primarily of collagen fibers
1. With minimal amounts of surrounding ground substance
2. Exhibit great tensile strength
3. Main function: support
4. Exhibit random orientation
5. Highly concentrated in areas that need strong support
o Except for Fibroblast
1. Other cells are sparse
Dense Regular Connective Tissue
o Present where great tensile strength is required
1. Ligaments and tendons
Attached to bones and are constantly subjected to strong pulling forces
o Collagen fibers
1. Parallel and dense in arrangement
Offer strong resistance to forces pulling along a single direction
Little ground substance is present
o Fibroblast
1. Predominant cell types
2. Located between rows of collagen fibers
 Ground substance in connective tissue
o Consists primarily of amorphous, transparent, and colorless extracellular matrix
o Has the properties of a semifluid gel
o High water content
o Contains different types of mixed, unbranched polysaccharide chains of:
Glycosaminoglycans, proteoglycans, adhesive glycoproteins
o Semifluid consistency
Facilitates diffusion of oxygen, electrolytes, nutrients, fluids, metabolites, and
other water soluble molecules
o Viscosity
Efficient barrier
Prevents movement of large molecules and spread of pathogens from the CT
into the bloodstream.
o Density
Depends on the amount of extracellular tissue fluid or water
Hyaluronic acid
o Constitutes the principal glycosaminoglycan of CT.
Proteoglycan aggregates
o Attract large amounts of water
Forms the hydrated gel of the ground substance
Hyaluronidase
o Enzyme that hydrolyzes hyaluronic acid
o Reduces viscosity of the ground substance
o Allow pathogens to invade the surrounding tissues
Mineralization
o Result of increased calcium deposition
o Changes density, rigidity, and permeability to diffusion
o Seen in normal developing cartilage models and bones
Adhesive glycoproteins
o Fibronectin
Adhesion protein
Binds connective tissue cells, collagen fibers, and proteoglycans
Interconnecting all three components of the CT
o Integrins
Integral proteins of the PM
Bind to extracellular collagen fibers and to actin filaments in the cytoskeleton
Established a structural continuity between the cytoskeleton and the ECM
o Laminin
Large glycoprotein
Major component of the cell basement membrane
Binds epithelial cells to the basal lamina
 Fibroblasts
o Dominant cells in the CT
o Highly active cells
o Irregular branched cytoplasm
o Synthesize collagen, reticular, and elastic fibers, as well as carbohydrates:
Glycosaminoglycans, proteoglycans, and glycoproteins
Fibrocytes
o Spindle shaped
o Smaller than fibroblasts
o Mature and less active cells
Macrophages or Histiocytes
o Phagocytes
o Ingest bacteria, dead cells, cell debris, and other foreign matter in the CT
o Antigen presenting cells to lymphocytes
o Derived from monocytes
o Liver- kupffer cells
o Bone- osteoclasts
o CNS- Microglia
Lymphocytes
o Most numerous cells in the Loose CT of the respiratory and gastrointestinal tracts
o Mediate immune responses to antigens by producing Abs
o Kill virus-infected cells by inducing apoptosis
Plasma cells
o Derived from lymphocytes
Exposed to antigens
o Synthesize and secrete antibodies
Destroy specific antigens
Adipose cells
o Store fat
o Provide protective packing material in and around numerous organs
Neutrophils
o Active and powerful phagocytes
o Engulf and destroy bacteria at sites of infections
Eosinophils
o Active and increase in number
After parasitic infections or allergic reactions
o Phagocytize Ag-Ab complexes formed during allergic reactions
Classification of CT

Connective tissue
o Develops from mesenchyme, an embryonic type of tissue
Present in the umbilical cord and in the pulp of developing teeth
o Consist of cells and extracellular matrix
o Extracellular Matrix
Consists of connective tissue fluid, ground substance
Embedded the different protein fibers (Collagen, elastic, reticular)
o Binds, anchors, and supports various cells, tissues, and organs of the body
o Loose CT or dense CT

Loose Connective tissue

Loose CT
o More prevalent in the body than dense CT
o Loose, irregular arrangement of CT fibers and abundant ground substance
o Fibroblast
Most common cell type
o Collagen fibers, fibroblasts, adipose cells, mast cells, and macrophages predominate

Dense Connective Tissue

Dense CT
o Contains thicker and more densely packed collagen fibers
o Fewer cell types
o Less ground substances\
o Fibroblasts
Most abundant cells
Dense irregular CT
o Collagen fibers
Random and irregular orientation
o Present in the dermis , capsules of different organs, and areas that need strong support
Dense regular CT
o Densely packed collagen fibers
Regular and parallel arrangement
o Tendons and ligaments

Cells of the CT

2 most common cell types

o Active fibroblasts
Fusiform shaped
Synthesize all of the CT fibers and the extracellular ground substance
 o Inactive or resting fibrocytes
Adipose cells
o Singly or groups
o Stores fat
o When predominate, adipose tissue
Macrophages or histiocytes
o Phagocytic cells
o Most numerous in loose CT
Mast cells
o Closely associated with blood vessels
o Widely distributed in the CT of the skin and in the digestive tract and respiratory organs
o Spherical cells
o Filled with fine, regular dark-staining and basophilic granules
Plasma cells
o Arise from the lymphocytes that migrate into the CT
Leukocytes
o Main function: defend the organism against bacterial invasion
Fibroblasts and adipose cells
o Permanent or resident CT cells

Type of Collagen Fibers

Collagen fibers
o Tough, thick, fibrous proteins
o Unbranched
o Most abundant fibers
Type 1 CF
o Dermis, tendons, ligaments, and bone
o Very strong and offer great resistance to tensile stresses
Type 2 CF
o Hyaline cartilage and elastic cartilage
o Provide resistance to pressure
Type 3 CF
o Thin, branching reticular fibers
o Form the delicate supporting network of:
Lymph nodes, spleen, and bone marrow
Type 4 CF
o Basal lamina of the basement membrane

Reticular fibers

Consist mainly of T3 collagen

Thin and form delicate network in:
 o Liver, lymph nodes, spleen, hemopoietic organs, and locations where blood and lymph
are filtered
Support capillaries, nerves, and muscle cells
Visible when stained with silver stain

Elastic Fibers

Thin, small branching fibers

Allow stretch
Less tensile strength than CF
Composed of microfibrils and elastin
Found in abundance:
o Lungs, bladder, and skin
Walls of large vessels
o Smooth muscle synthesizes the elastic fibers.
Bilirubin

The reaction of bilirubin with a diazotized sulfanilic acid soln to form a colored product
o First described by Erlich in 1883 using urine samples
Classic diazo reaction
o Bilirubin with a diazotized sulfanilic acid soln
o Reaction on which all commonly used methods today are based
1913
o Van den Bergh
Found that the diazo reaction may be applied to serum samples
Only in the presence of an accelerator
1937
o Malloy and Evelyn
Developed the first clinical useful methodology for the quantitation of bilirubin
in serum samples
Using the classic diazo reaction with a 50% methanol solution as an
accelerator
1938
o Jendrassik and Grof
Described a method using the diazo reaction with caffeine-benzoate-acetate as
an accelerator
Unconjugated bilirubin= total bilirubin conjugated bilirubin
Bilirubinometry
o Used in quantifying bilirubin in the neonatal population
o Not applicable to adults
Carotinoid compounds causes strong positive interference
o Involves the measurement of reflected light from the skin using 2 wavelengths
Provide a numerical index based on spectral reflectance
o Microspectrophotometers
Determine the optical densities of bilirubin, hemoglobin, and melanin in the
subcutaneous layers of the infants skin
Unconjugated Bilirubin
o Non polar
o Water-insoluble substance
o Found in plasma bound to albumin
o Only react with the diazotized sulfanilic acid solution in the presence of an accelerator
(solubilizer)
Conjugated Bilirubin
o Polar
o Water-soluble compound
o Found in plasma in the free state (not bound to any protein)
o Reacts directly with diazo reagent without an accelerator
 Delta Bilirubin
o Conjugated bilirubin covalently bound to albumin
o Seen only in hepatic obstruction
o React in most laboratory methods as conjugated bilirubin
o Attached to albumin
Too large to be filtered by the glomerulus and excreted in the urine
3 fractions of total bilirubin:
o Conjugated, unconjugated, and delta bilirubin
3 fractions are known as total bilirubin

Specimen Collection and Storage

Total B methods using a diazotized sulfanilic acid

o Performed on either serum or plasma
o Serum
Preferred for the Malloy-Evelyn procedure
Fasting sample is preferred
o Lipemia- increase measured B concentrations
Avoid hemolyzed sample
o If hemolyzed, decreases the reaction of bilirubin with the diazo reagent
Should be protected from light
o If unprotected, B values may reduce by 30%-50% per hour
Stable for 2 days at RT, 1 week at 4 degree C, and indefinitely at -20 degree C
o If serum or plasma is separated from the cells and stored in the dark

Methods

Modified Jendrassik-Grof method using Caffeine-Benzoate as a solubilizer

o Candidate reference method for Total B
Jendrassik-Grof or Malloy-Evelyn
o Most frequently used method to measure bilirubin
Jendrassik-Grof advantages over the M-E method
o Not affected by pH changes
o Insensitive to a 50-fold variation in protein concentration of the sample
o Maintain optical sensitivity even at low bilirubin concentrations
o Minimal turbidity and a relatively constant serum blank
o Not affected by hemoglobin up to 750 mg/dL

Malloy-Evelyn Procedure

Bilirubin pigments are reacted with a diazo rgt

Diazo rgt reacts at the central methylene carbon of bilirubin to split the molecule forming two
molecules of azobilirubin
 pH- 1.2
o Azobilirubin produced is red purple in color
o Maximal absorption of 560 nm
Methanol
o Most commonly used accelerator to solubilize unconjugated B

Jendrassik-Grof for Total and Conjugated B Determination

B pigments in serum or plasma

o Reacted with a diazo reagent (sulfanilic acid in HCL and sodium nitrite)
Production of purple product azobilirubin
Individual fractions
o Determined by taking 2 aliquots of sample
Reacting 1 aliquot with the diazo rgt. Only
Yield conjugated B only
Other aliquot with the diazo rgt. and caffeine-benzoate accelerator
Solubilize the water-insoluble fraction of bilirubin
Yield a total bilirubin value
o Reaction of aliquots with the diazo rgt.
Terminated by Ascorbic acid
Destroys the excess diazo rgt.
Alkaline tartrate solution
o Alkalinizes the solution
o Shifts the absorbance spectrum of the azobilirubin to a more intense blue color that is
less subject to interfering substances
Final blue product
o Measured at 600 nm with the intensity of color produced directly proportional to B
concentration
Comments and sources of error
o Standardized instrument
Maintain reliable B results
o Careful preparation of B standards
Critical as these are subjected to detection from exposure to light
o Hemolysis and lipemia should be avoided
Alter B concentration
Activators
o Metal ions
Fe, Mn, Co, Cu, Zn, Se, and Mo
Coenzyme
o Important coenzymes
NADH, FADH, Quinone, CoA
o Common coenzymes
 Vitamin A, Vitamin C, Vitamin B1,B2,B3,B6 and B12, Vitamin H, Vitamin K
Zymogen
o Angiotensinogen, trypsinogen, chymotrypsinogen, pepsinogen, fibrinogen
Isoenzyme
o LDH1, LDH2, LDH3, LDH4, LDH5
o CK1, CK2, CK3
Apoenzyme
o Requires a cofactor but does not have one bound
Holoenzyme
o Apoenzyme together with its cofactor
o Examples: DNA polymerase, RNA polymerase
EC numerical code
o First digit- class
o Second and third digit- subclass and subsubclass of the enzyme
o Fourth digit- serial number specific to each enzyme in a subsubclass
Metalloenzyme
o Carbonic anhydrase, Vitamin B-12 dependent enzymes (Cobalamin)
o Nitrogenase
o Superoxide dismutase
ES complex
o Physical binding of the substrate to the active site of an enzyme
Absolute specificity
o Enzyme combines with only 1 substrate and catalyzes only the one corresponding
reaction
Group specificity
o Combine with all substrates containing a particular chemical group
Phosphate ester
Bond Specificity
o Specific to chemical bonds
Stereoisometric specificity
o Enzymes that predominantly combine with only one optical isomer of a certain
compound
First order kinetics
o Reaction rate is directly proportional to substrate concentration
Zero-order kinetics
o Reaction rate depends on enzyme concentration
Michaelis-Menten constant (Km)
o Constant for a specific enzyme and substrate under defined reaction conditions and
o Is an expression of the relationship between the velocity of an enzymatic reaction and
substrate concentration
 o Specifically the substrate concentration at which the enzyme yields half the possible
maximum velocity
o Indicates the amount of substrate needed for a particular enzymatic reaction
Activators
o Enhance the reaction rate in proportion with concentration
o Function by alternating the spatial configuration of the enzyme for proper substrate
binding, linking substrate to the enzyme or coenzyme, or undergoing oxidation or
reduction
Coenzymes
o Second substrate for enzymatic reactions
o If increased, increase the velocity of an enzymatic reaction in a manner synonymous
with increasing substrate concentration
With a substrate conc. significantly higher than the conc. of inhibitor
o Inhibition is reversible
o Competitive inhibition
Uncompetitive inhibition
o Increasing substrate conc. increases inhibition
2 general methods to measure the extent of an enzyme activity
o Fixed-time
Reactants are combined
The reaction proceeds for a designated time
The reaction is stopped (usually by inactivating the enzyme with a weak acid),
A measurement is made of the amount of reaction that has occurred
The reaction is assumed to be linear over the reaction time
The larger the reaction, the more enzyme is present
o Continuous monitoring or kinetic assays
Multiple measurements, usually of absorbance change, are made during the
reaction, either at specific time intervals (usually every 30 or 60 seconds)
Or continuously by a continuous-recording spectrophotometer
Advantageous over fixed-time mtds
Linearity is readily observable
Most common cause of deviation from linearity
o Enzyme is so elevated that all substrate is used early in the reaction time
Activity units
o Units used to report enzyme levels
International unit
o The amount of enzyme that will catalyze the reaction of 1 micromole of substrate per
minute under specified conditions
Enzymes are usually expressed in units per liter
Katal
o International system of unit
 o Mol/s
Mol-unit for substrate conc.
S- second
Immunoassay methodologies
o Quantify enzyme concentration by mass
o Routinely used for quantification of some enzymes such as (CK)-MB
Electrophoretic techniques
o Provide resolution of isoenzymes and isoforms
Clinical Laboratory Improvement Amendment of 1988
o Established guidelines for quality control and proficiency testing for all laboratories