FINALS CLINICAL CHEMISTRY a.

globular: relatively symmetrical with compactly folded and

coiled polypeptide chains
PROTEINS b. fibrous: more elongated and asymmetrical and have a
PROTEINS higher viscosity
● Biochemical essential for the body
2. CONJUGATED
STRUCTURE AND PHYSIOLOGY ● Protein (apoprotein) + nonprotein (prosthetic group)
● Proteins are MACROMOLECULES; they have a large ● Has 3 chemical composition of protein
structure as they are composed of linked polymers of ● Lipid content is present
amino acids
● Covalently linked polymers of amino acids Prosthetic group (3 chemical composition of protein):
● Amino acids are linked via PEPTIDE BOND (formed - Lipid (lipoprotein)
upon removal of a water molecule) - carbohydrate (glycoprotein)
● Amphoteric - metals (metalloprotein)
- Can be polar or nonpolar
- NonPolar: no charge
- Polar: Acidic (negative charge) or Basic FUNCTIONS
(positive charge) - Repair body tissues
- Polar Neutral: Can either be positive or - Important in blood coagulation and immunologic
negative function
● Synthesized in the liver & secreted by the hepatocyte - For transport of metabolic substances
into the circulation EXCEPT immunoglobulins - Maintenance of osmotic pressure
(plasma cells) - Maintenance of blood pH (buffers)
● Provide 12 – 20% of the total daily body energy - Biocatalysts: Induce metabolism, CATABOLISM
requirement specifically
● Composes 50 – 70% of the cell’s dry weight
*In the formation of blood, coagulation proteins are highly
AMINO ACIDS needed
- Secreted by HEPATOCYTES (cells in the liver) METABOLISM
- Synthesized in the LIVER 1. Dietary Intake
- There are 20 ESSENTIAL amino acids 2. Absorption
3. Production
PROTEIN VS. POLYPEPTIDE 4. Storage
- Protein and Polypeptide can be interchangeable 5. Destruction

4 STRUCTURES MAJOR PLASMA PROTEINS

1. PRIMARY STRUCTURE 1. PRE-ALBUMIN (Transthyretin)
● Sequence of the amino acids in the polypeptide ● Migrates ahead of albumin
chain ● Rich in tryptophan and contain 0.5% carbohydrate
● Analytical processes: chromatography, ● Serves as transport protein for T3, T4 and retinol
electrophoresis, dye binding and light absorbance (Vit. A)
depend on this sequence RETINOL
● Purest form of Vitamin A
2. SECONDARY STRUCTURE
● Arises from the interaction among the different RETINOID
segments of a polypeptide chain ● Type of retinol

Three structures: Electrophoresis

a. alpha-helix – chain forms a regular helix ; coil resembling a - Involves anodes (+) and cathodes (-)
spring - Protein is a cathode, therefore it migrates in the
b. beta-pleated sheets – in fully extended structures; flat, anode
corrugated structure - Follows the principle of: Opposite Attracts
c. random coils – no apparent pattern
Increased: alcoholism, chronic renal failure, steroid
3. TERTIARY STRUCTURE treatment
Decreased: poor nutrition, liver disorder, malignancy
● actual 3-dimensional structure or folding pattern of
the protein
2. ALBUMIN
4. QUATERNARY STRUCTURE ● Largest plasma protein fraction (52-62%)
● association of several polypeptide chains into larger ● Synthesized in the liver at a rate that is dependent on
“oligomeric” aggregate unit protein intake
● Stable complexes: dimers, trimers, tetramers ● Serves as circulating reservoir of amino acids
● Regulator of osmotic pressure
Classification ● Transport protein because of ease of binding with
1. SIMPLE blood components
● Contain peptide chains that on hydrolysis yield only ● “negative acute phase reactant”
AA ● Sensitive & highly prognostic marker in cases of
● AMINO ACID ONLY! cystic fibrosis
Reference values: 3.5 – 5.0 g/dL (35 – 50 g/L)
● Increased albumin (Hyperalbuminemia):
- Hemoconcentration
- Dehydration
- Excessive albumin infusion
● Decreased albumin (Hypoalbuminemia):
- Decreased synthesis (liver impairment)
- Malabsorption or malnutrition
- Nephrotic syndrome (renal loss)
- Severe burns
* Albumin levels depends on the diet intake
3. GLOBULINS
● Heterogenous complex mixture of protein molecules
(α1, α2, β and γ fractions)
● Elevated concentration of globulin in early cirrhosis
(occurs in the liver) will balance loss of albumin
resulting to normal levels of total protein
- normal A/G ratio : 1.3 – 3 : 1
- low A/G ratio – liver diseases, infectious diseases, multiple
myeloma, nephritis
*Albumin/Globulin (A/G) A- 1.3 G- 3:1
Alpha1-globulins
a. α-1-antitrypsin
● 90% of the α1-globulin band
● Acute phase reactant
● Neutralize trypsin-like enzymes
● Major inhibitor of protease activity; inhibit lysosomal
elastase released from PMNs during their response to
particles & inhaled bacteria
● Major liver involvement
Increased: inflammation, pregnancy and contraceptive use
Decrease: associated with emphysematous pulmonary
disease & juvenile hepatic cirrhosis
b. α-1-fetoprotein
● Synthesized initially by the fetal yolk sac & then the
parenchymal cells of the liver
● Used as a tumor marker (hepatic & gonadal CA)
● Fetal involvement
● Screening test for any fetal conditions, increase
passage of fetal proteins into the amniotic fluid;
detects neural tube defects
Increased: neural tube defects (spina bifida), atresia of the
GIT, fetal distress, ataxia telangiectasia, tyrosinosis,
hemolytic disease of the newborn (HDN)
Decreased: Down's syndrome
c. α-1-Acid Glycoprotein (orosomucoid)
● Contains high percentage of CHO and sialic acid
(45% CHO + 11-12% Sialic acid)
● Synthesized both by the liver & by granulocytes and
monocytes (WBCs)
● Inhibits the phagocytic activity of neutrophils &
inhibits platelet aggregation
● May inactivate progesterone
● Presence of this affects blood coagulation
Increased: pregnancy, cancer, pneumonia, rheumatoid
arthritis (RA), cell proliferation
Decreased: nephrotic syndrome
d. α-1-Antichromotrypsin
Alpha2-globulins
A. HAPTOGLOBIN
● An acute phase reactant
● Synthesized in the hepatocytes & cells of the RES
● Binds free hemoglobin by its α- chain
● One of the proteins used to evaluate rheumatic
diseases
Increased: inflammation, nephrotic syndrome, burns,
trauma
Decreased: intravascular hemolysis and liver disease
B. CERULOPLASMIN
● Copper-containing protein BUT does NOT transport
copper; synthesized in the liver, where 6-8 copper
atoms are attached
● Serves as an antioxidant to prevent lipid peroxidation
& cellular damage
● Increased in inflammation and pregnancy
● Indicator for Wilson's disease (0.1 g/L)
● COMPLEX
● Does not transport copper, it only syntheize it
Decreased: Wilson’s disease, malnutrition, malabsorption,
nephrotic disease, Menke’s disease (kinky hair syndrome)
a. α-2-Macroglobulin
● Largest major non-immunoglobulin protein in plasma
● Found principally in the intravascular spaces; does
not diffuse from the plasma space
● Lower amounts can also be found in CSF
● Inhibits proteases such as trypsin, pepsin and
plasmin (3) Beta-globulins
Beta Globulins
a. Transferrin (Siderophilin)
● Major component of the β2-globulin fraction
● A glycoprotein synthesized in the liver
● Tansports oxidized iron (Fe+3) to its storage sites
● Prevents loss of iron through the kidney
Increased: hemochromatosis (bronze skin)
Decreased: liver disease, malnutrition, nephrotic syndrome
b. β2-Microglobulin
● Light chain component of the major human leukocyte
antigen (HLA)
● Found on the surface of most nucleated cells
● Present in high concentration on lymphocytes
● Filtered by the renal glomerulus but reabsorbed
LYMPHOCYTES
- Indicator for viral infections
- ↑ Viral infection ↑ B2-Microglobulin
Increased: RA, systemic lupus erythematosus (SLE), HIV
5 IMMUNOGLOBULINS (GAMED)
1. IgG
2. IgA
3. IgM
4. IgE
5. IgD
4. GAMMA-GLOBULINS
Increased in
- chronic inflammation
- cirrhosis or viral hepatitis
 - collagen diseases
- paraproteins (monoclonal bands, gammopathies)
Decreased in
- congenital or acquired immunodeficiency
- Immunoglobulins – synthesized in the plasma cells
- not produced to any extent by the neonate
5. FIBRINOGEN
● One of the largest proteins in the blood plasma
● Synthesized in the liver
● Most abundant of the coagulation factors
● An acute phase reactant; markedly increased during
inflammatory process
● May serve as a marker for long-term prognosis of
cardiovascular disease
● Distinct band between β and γ-globulins on
electrophoresis
● Forms a fibrin clot when activated by thrombin
● Last protein to be activated to have a stable clot
● AKA FACTOR 1
FIBRIN
- Activated fibrinogen
THROMBIN
- Activates fibrinogen to become fibrin
Increased: pregnancy and use of birth control pills
Decreased: extensive coagulation
Reference values: 200 – 400 mg/dL (2.0 -4.0 g/L)
6. LIPOPROTEIN
● Complexes of proteins & lipids (LDL, HDL, VLDL,
Chylomicrons)
● Transports cholesterol, triglyceride and phospholipids
● Transports protein in general
Low Density Lipoprotein (LDL)
- Bad cholesterol in lipid profile test
- Clogs or blocks normal passage of blood in the heart
which causes a heart attack
High Density Lipoprotein (HDL)
- Good cholesterol in lipid profile test
Very Low Density Lipoprotein (VLDL)
*In the lipid profile test these are tested:
- Cholesterol
- HDL
- LDL
- Triglyceride
7. COMPLEMENT
● Participates in the immune reaction and serve as a
link to the inflammatory response
● Circulates as non-functional precursors
Increased: inflammatory states
Decreased: SLE, DIC, malnutrition
8. C-REACTIVE PROTEIN
● Appears in blood of patients with diverse
inflammatory diseases
● Undetectable in healthy individuals
● Precipitates with the C substance, a polysaccharide of
pneumococci
● General scavenger molecule; gamma-migrating
protein
Increased: acute rheumatic fever, MI, RA, gout, bacterial &
viral infections
MISCELLANEOUS PROTEINS
1. MYOGLOBIN
● A heme protein or oxygen-binding transport protein
found in skeletal and cardiac muscles
● Approximately 2% of the total muscle protein
● Marker for chest pain (angina) and early detection of
acute myocardial infarction (AMI)
● In AMI, the onset is 1-3 hours, peak level 5-12 hours,
normalize in 18-30 hours
● Useful marker for monitoring the success or failure of
reperfusion
Increased: AMI, angina, rhabdomyolysis, muscle trauma,
extraneous exercise, IM injection
2.TROPONINS
● A complex of 3 proteins that bind to the thin filaments
of striated muscle (cardiac & skeletal)
● Diagnostic marker for identifying cardiac injury in the
presence of a skeletal muscle damage
● Levels in blood may elevate after AMI in the absence
of CK-MB elevations
TnT, TnI, TnC = muscle contraction
3.AMYLOID
● A pathological extracellular deposit associated with a
group of disorders collectively called amyloidosis.
MEASUREMENT OF PROTEINS
1. KJELDAHL METHOD
● Determination of protein nitrogen derived from
constituent amino acids
● Nitrogen in pff is converted to ammonia using H2SO4
◾
⦿ The nitrogen in ammonia may be measured using:
◾ Nesslerization
◾ Berthelot reaction
Titration method
2. BIURET METHOD
● Copper binds to the peptide bond structure of the
protein, forming a purple-colored chromogen
3. FOLIN-CIOCALTEU METHOD
● Tyrosine & tryptophan in proteins reduce PT-PMA
reagent (folin-ciocalteu) to give a blue color
● Detects proteins in concentrations as low as 10 – 60
μg/mL
● Widely used in research to measure tissue & enzyme
proteins
4. Absorption of UV light at 280 nm
● Chiefly the result of the high electron density of
aromatic rings of tyrosine & tryptophan in solution (pH
8)
5. Dye-Binding Methods
● Based on the ability of proteins to bind certain dyes
● Bromocresol Green Method (BCG) for Albumin
● Used to stain protein bands after electrophoresis (e.g.
Coomassie Brilliant Blue)
6. PROTEIN ELECTROPHORESIS 3 COMPONENTS OF AMINO ACIDS
● Direction of migration of proteins in an electrical field 1. Amino Group/ Amine Group: Contains Nitrogen
determined by surface charge & MW of protein 2. Carbon Structure
● Urine protein electrophoresis exactly same as serum 3. Carbonyl group: COOH
except it must be concentrated before application : Also called as Carboxyl Acid
● Support media include cellulose acetate, agarose gel
and starch gel Amine + Carboxyl Acid = Amino Acid
STAINS: Amino acid + Amino acid + Amino acid = Set of amino acid
- amido black chain
- ponceau S and coomassie brilliant blue ● Connected through the end of amino group and
another end of amino group
SIGNIFICANT FINDINGS: ● When 2 amino acids are linked or joined together,
❖ Beta-gamma bridging effect: due to IgA (in serum); there is a removal of water, thus creates PEPTIDE
Cirrhosis BONDS
❖ Monoclonal band (gamma-globulin): Monoclonal ● PEPTIDE BONDS: link that binds 2 amino acids
gammopathy (Multiple myeloma) ● If there are many amino acids that are linked together
❖ Polyclonal band: Chronic inflammation it is called a POLYPEPTIDE BOND (PROTEIN)
❖ Increase in α-2-macroglobulin: nephrotic syndrome ● Protein and Polypeptide are used interchangeably
❖ α-1-globulin flat curve: deficiency in α-1-antitrypsin; ● There are 20 essential amino acids
Juvenile cirrhosis ● Amino acids can be classified according to their
FUNCTION
AMINO ACIDURIA
1. OVERFLOW GLUCOGENIC AMINO ACID
● Plasma level above renal threshold as a result of 1. Alanine
metabolic disorder 2. Arginine
3. Aspartate
a. Phenylketonuria (PKU) ➔ Involved in Krebs Cycle
● Enzyme deficiency (phenylalanine hydrolase) which ➔ Involved in carbohydrate metabolism
leads to :
- increased phenylalanine in the blood KETOGENIC AMINO ACID
- phenylpyruvic acid (prime metabolite) is 1. Leucine
present in both blood and urine in elevated 2. Lysine
concentration ➔ Generates KETONE BODIES
b. Branched chain ketoaciduria - FRUITY ODOR if Ketonuria is present
● Maple syrup urine disease - Acetoacetic Acid
● Caused by markedly reduced or absence of α-keto - Beta Hydroxybutyric Acid
acid decarboxylase - Acetone
● Increased branched chain amino acids (leucine, - These 3 are found in the urine indicating
isoleucine & valine) in blood, urine and CSF Ketonuria

2. RENAL BOTH KETOGENIC AND GLUCOGENIC AMINO ACID

● Normal plasma level but decreased renal threshold or 1. Isoleucine
reabsorption 2. Phenylalanine
cystinuria – increased cystine, lysine, ornithine, arginine in 3. Tryptophan
urine 4. Tyrosine
5. Threonine
METHODS
1. Screening tests ESSENTIAL AMINO ACID
a. TLC with ninhydrin 1. Arginine
b. urine color tests 2. Histidine
c. Guthrie microbiological tests : PKU 3. Isoleucine POLAR BASIC AMINO ACID
4. Leucine
2. Quantitative tests 5. Lysine
a. Ion-exchange chromatography 6. Methionine
b. HPLC 7. Phenylalanine
c. GC-MS 8. Threonine
9. Tryptophan
SHAPE OF PROTEINS 10. Valine
1. Fibrous: Elongated ➔ Not produced in the body
2. Globular: Symmetrical ➔ Supplemented in by the diet/ food intake

CHEMICAL COMPOSITION OF PROTEINS NON ESSENTIAL AMINO ACID

1. Simple: Has amino acids 1. Alanine
2. Conjugated/ Complex: Protein+NonProtein 2. Asparagine
3. Aspartic Acid POLAR ACIDIC AMINO ACID
AMINO ACIDS 4. Cysteine
● Building blocks of protein 5. Glutamic Acid
6. Glutamine
 7. Glycine ● Absorbed during digestion
8. Proline
9. Serine Glutamic Acid
10. Tyrosine ● Present in variety of foods (Umami)
● Transports Potassium into the spinal fluid
COMMON AMINO ACID ● Potassium transport
1. Glutamine
2. Leucine Glutamine
● Transports ammonia to urea
ESSENTIAL AMINO ACIDS
Arginine Glycine
● Production of Urea ● Simplest amino acid
● Synthesized from SERINE
Histidine
● Direct precursor of histamine Proline
● Helps grow and repair body tissues ● Derived from GLUTAMINE

Isoleucine Serine
● Formation of Hemoglobin
● Regulation of glucose ● Component of myelin sheath
● Derived from 3-PHOSPHATE IN GLYCOLYSIS
Leucine ● Involved in lipid and fatty acid metabolism: creatine &
● Helps in nitrogen balance in adults porphyrins
● Helps in the optimal growth in infants
● SECOND MOST COMMON AMINO ACID Tyrosine
● Produced from PHENYLALANINE
Lysine ● Precursor for hormones
● Production of antibodies ● ↓Phenylalanine= ↓ Tyrosine
● Formation of collagen ● Precursor of T4 (Thyroxine)
● Lowers triglyceride levels - Hormone in the thyroid gland

Methionine
● Initiates translation of mRNA
● Helps in the breakdown of fats

Phenylalanine
● Production of norepinephrine
● Used to treat arthritis and depression
● Component of natural sweetener ASPARTAME
- Aspartame: artificial sweetener made up of
two amino acids
1. ASPARTIC ACID
2. PHENYLALANINE

Threonine
● Formation of collagen, elastin, and tooth enamel

Tryptophan
● Treatment of migraine headaches
● Controls hyperactivity in children

Valine
● Treatment for insomnia and anxiety
● Helps in nitrogen balance in adults

NON ESSENTIAL AMINO ACIDS

Alanine
● Transfer protein of Nitrogen from tissues to liver
● Product of the breakdown of DNA

Asparagine
● Production of ammonia
● Important role in the synthesis of ammonia

Aspartic Acid
● Involvement in gluconeogenesis
● Metabolite in the citric acid cycle and the urea cycle

Cysteine