Clinical Chemistry 1

Protein
Classification
Introduction By Functions: By Structure:
- Serve a key role in transport, synthesis, storage Enzymes Simple Proteins
and clearance of substances. - Catalyze - Composed of only
- Some Major role: chemical amino acids.
 Catalyzing biochemical reactions by reactions - Globular:
enzymes. - Mostly of o Globe like,
 Provide structure and support the cell enzymes are symmetrical
(e.g collagen) intracellular. proteins that is
 Transport of materials (e.g Transferrin) - If they are ↑ in soluble in water.
 Participates in immune response as concentration o Example:
antibodies. signifies that  Albumin
Structure there are - Fibrous:
Primary - Refers to an amino problem in o Structural proteins
acid in the specific certain organ in connective
sequence tissues, tendons,
- Linear muscle and bone
- Composed of o Example:
peptide bonds  Troponin and collagen
- Involved in post- Hormones Conjugated Proteins
translational - Control the - Consist of protein and
modifications actions of cells non-protein
Secondary - Formed by folding in organs (prosthetic) group.
of peptide chains - Chemical - Examples:
to an alpha-helix or messenger  Metalloprotein
beta-sheet - Affect growth, o Metal ions
- Regularly development, (e.g.
repeating structure metabolism of Ceruloplasmin-
- Stabilized by cells transport
hydrogen bonds - Example: copper)
and peptide bonds  Insulin o Complex
- Examples: Transport metal:
 Collagen - Transport ions Hemoglobin
 Actin and molecules  Lipoproteins
 Myosin across o Lipids (e.g. LDL,
 Keratin membrane HDL, VLDL)
 Elastin Immunoglobulins  Glycoproteins
Tertiary - Refers to the 3D - Mediates o ↓ carbohydrate –
structure of a immune (e.g. Haptoglobin,
single protein response alpha1-
molecule - Produced by b antitrypsin)
- Alpha and beta cells (plasma  Mucoproteins of
complexes are cells) proteoglycans
folded into Structural o ↑ Carbohydrate
compact globule - They give form (e.g. mucin)
due to hydrophobic in cells and  Nucleoprotein (e.g.
interaction tissues. chromatin)
- There are disulfide Storage
bridges and ionic - Proteins can
bonds also serve as
- There are fibrous reserve for
and globular metal ions and
structures. amino acids.
Quaternary - Interaction of more - Example:
than one protein  Ferritin
molecule or Energy source
subunits - Reserve
- Example: source of
 Hemoglobin energy

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Clinical Chemistry 1
- Example: Albumin Globulin
 Creatine Pre-albumin Alpha1-Globulins
Osmotic force (transthyretin)  α1 – Antitrypsin
- Distribution of - Pre-albumin - protease inhibitor
water in because it (neutrophil
components of migrates before elastase) –
the body the actual albumin secreted by
- Proteins exert - Binds thyroid leukocytes when
colloid osmotic hormones (T3 and there is infection.
force. T4) Cannot distinguish
Hemostasis - Binds to retinol- properly that can
- Coagulation in binding protein lead to damage
blood (transport Vit. A) that is why they
- Coagulation - ↓ level of pre- inhibit.
factors are albumin can be - acute phase
made up of seen in hepatic reactant (+)
proteins damages
Acid Base Balance - Seen also in acute  α1- Fetoprotein
- Buffers to phase - principal fetal
blood maintain inflammatory protein
pH response. - ↓ levels can be
- Protein buffer - Poor nutritional seen in down
system—most status syndrome,
abundant - ↑ levels can be Trisomy-18
buffer in intra seen in patients (Edward
and receiving steroid syndrome)
extracellular therapy, alcoholics, - ↑ levels in fetal
fluid. and chronic renal distress, spina
Note: failure bifida,
- The carboxyl end of amino acids can act as - Negative Acute anencephaly
acid and can release hydrogen when pH rises Phase Reactant - Should not be
- Amino end can act as base by combining with seen in adult;
hydrogen when pH falls when seen
indicator that there
is hepatocellular
Plasma Proteins carcinoma in the
patient.
 α1 – Acid
glycoprotein
- regulates immune
system
- APR
 α1 - Lipoprotein
- transport lipids,
cholesterol (HDL)
- marker for Acute
Coronary
Syndrome (↑)
 α1 –
Antichymotrypsin
- serine proteinase
inhibitor
- cleave enzyme to
inactivate
- mutations can be
seen in patients
with Parkinson’s
and Alzheimer
disease, APR
 Inter-α-trypsin
inhibitor

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- Serine proteinase - ↑ levels during  α2 –
inhibitor dehydration of Macroglobulin
- APR patients. - protease inhibitor
 Gc-globulin - 10x ↑ in
- Group specific Nephrosis—
globulin degenerative
- Transports Vit D disease in kidney
and binds actin tubules.
(from injured cells) - ↑ liver disease,
- ↓ levels seen on diabetes, user of
liver disease, oral contraceptive
tissue injury. Beta- Globulins
 Pre-ß-lipoprotein
- Transport lipids,
triglyceride (VLDL)
- Marker for acute
coronary syndrome
 ß-Lipoprotein
- trans port
cholesterol (LDL)
- marker for ACS
 Transferrin
(Siderophilin)
- Transport iron to
apoferritin & BM
Hemosiderin-- ↑
iron (iron
precipitate in
tissue)
- (-) APR ↓,
Hemochromatosis
(iron deposits)
Albumin Alpha2 Globulins - ↑ level in IDA
- Most abundant  Haptoglobin  Hemopexin
protein in the - Binds to - Transport heme
plasma. hemoglobin to from circulation to
- Can also be seen prevent loss of liver to recycle iron
in tissues constituent of - APR
- Major transporter hemoglobin into - ↓ Hemolytic
of unconjugated urine like iron. anemia
bilirubin (B1), fatty - ↓ low levels can be - ↑ inflammation,
acids, steroids, seen in hemolytic diabetes, muscle
electrolytes (Ca2+, disease (HTR, dystrophy
Mg2+; T3 & T4) HDN)  ß2- Microglobulin
- Contributor to - APR - component of HLA
oncotic pressure— in nucleated cells
humihila ng tubig  Ceruloplasmin - ↑ seen in impaired
papunta sa kaniya. - Binds 90% of kidney function,
- ↓ nephrotic copper overproduction of
syndrome, burns - ↓ level seen in protein
- Analbuminemia Menkes syndrome  C4, C3, C1q
(absence of (kinky hair - Part of humoral
albumin) disease), Wilson’s nonspecific
- Bisalbuminimia (2 disease (↑ Cu in immune response
bands) brain & Liver), - APR
- ↓ level in liver Kayser-Fleischer - C3 most abundant
disease (cirrhosis), rings (cornea) followed by C4
GI loss (diarrhea) - APR - ↑ seen in acute
inflammatory

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Clinical Chemistry 1
disease, tissue Fetal Fibronectin - Placental
inflammation adherence to the
- ↓ level of C3 seen uterus
in autoimmune - If the age of
diseases and gestation is
infections. between 22-36
 Fibrinogen weeks and fetal
- Precursor of fibrin fibronectin is seen
clot there is a high
- Largest protein in chance of preterm
blood plasma labor and
- (+) APR delivery.
 C-Reactive Adiponectin - Hormone from
Protein adipocytes
- Opsonin (promotes - ↓ level associated
phagocytosis) with heart
- APR (rheumatoid diseases, Type 2
arthritis, SLE) ↑ Diabetes Mellitus
Gamma-Globulins and Obesity
 IgG Cross-linked C- - Proteolytic
- Most abundant Telopeptides fragment of
 IgA collagen I—during
- For secretions bone turnover
 IgM - Marker for bone
- Early response resorption
 IgE ß-Trace Protein - Prostaglandin D
- Allergy response synthase
 IgD - Marker for CSF
- surface leakage
- Potential marker
Other Plasma Proteins for kidney function
Myoglobin - globular protein - Not affected by
with heme glucocorticoid
- oxygen carrier in therapy unlike
muscles (skeletal Cystatin C.
and cardiac) Cystatin C - Cysteine
- ↑ level can be seen proteinase inhibitor
in AMI, Muscular - Marker for kidney
dystrophy, function (GFR)↑
Crushing injury Amyloid - Fibrous protein
Troponin (cTn) - Structural protein - ↑Amyloid ß42=
- “Gold standard” in Alzheimer disease
suspecting AMI.
Brain Natriuretic - Hormone that Total Protein Abnormalities
Peptide regulates body - In laboratory if we measured total protein, what
fluid homeostasis we measure is all the plasma proteins
and blood pressure - Total protein is used to evaluate various
- Regulates by conditions such nutritional status of the patient.
natriuresis— - ↓ level when there is not enough intake, or
excrete sodium in essential amino acids going to the body.
urine and diuresis - Can also be used in detecting kidney diseases
– excrete water in such as nephrotic syndrome.
urine. - Can also detect liver disease.
- By ↓ angiotensin II - Total Protein 5.7-9 g/dL:
and ↓ - 53-65% (3.5-5 g/dL) = Albumin
norepinephrine - 35-47% (2.2-4 g/dL) = Globulin
synthesis.
- Marker for
Congestive heart
failure

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3 Major Steps:
Hypoproteinemia 1. Digestion
Disease: TP Albumin Globulin 2. Distillation
Hepatic Damage N, ↓ ↓ ↑ 3. Titration
 Cirrhosis ß-ɣ Refractometry - Measurement of
bridging refractive index
 Hepatitis ↑ ɣ- due to solutes in
globulins serum
 Burns, Trauma - Use in estimate
Burns, Trauma N, ↓ ↓ ↑ plasma protein.
Infections Biuret - Most widely used
N, ↓ ↓ ↑
method for
 Acute- ↑ α1, α2
determination of
globulins
TP.
 Chronic - ↑ α1, - In alkaline medium
α2, ɣ-globulins and in the
Malabsorption ↓ ↓ N presence of at
(Sprue) and least 2 peptide
Inadequate diet bonds, Cu2+ ions
Nephrotic ↓ ↓ N will complex with
syndrome group involve in
- ↑α2 the peptide bond to
(macroglobulin), form pink- reddish
ß globulins violet chelate.
- ↓ ɣ globulins - Measured using
Immunodeficiency ↓ N ↓ spectrophotometer
Syndrome at absorbance of
Salt retention ↓ ↓ ↓ 540 nm
syndrome Dye binding - Protein binds to
- Patient do not  Bromphenol blue dye and causes a
excrete sodium  Ponceau S spectral shift in the
 Amido Black 10B absorbance
Hyperproteinemia  Lissamine green
Diseases TP Albumin Globulin  Coomassie brilliant
Dehydration blue
↑ ↑ ↑
 Vomiting
 Diarrhea Albumin (A) & Globulin (G)
 Diabetic acidosis Salt precipitation - Globulins are
 Monoclonal & ↑ N ↑ separated from
Polyclonal albumin by
Gammopathy precipitation
 Multiple myeloma process using Na
 Waldenstrom’s Salt, then albumin
macroglobulinemia in supernatant is
quantitated by
Method of Analysis biuret.
Dye Binding - Most widely used
Specimen Consideration - Concentration of
- Fasting specimen is NOT needed albumin is
- Interferents are lipemia and hemolysis (↑ TP) calculated by
- Reference interval: measuring the
o 6.5-8.3 g/dL = ambulatory absorbance of the
o 6.0-7.8 g/dL = recumbent albumin dye
complex which is
proportional to the
Total Protein
specimen albumin
Kjeldahl - Reference method
concentration.
- Assume average
Methyl Orange - Nonspecific for
content of 16%
Albumin
- Time consuming
HABA - Interference
(salicylates,bilirubin)

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BCG (Bromcresol - Sensitive
green) - Most commonly
used Serum or Plasma + Biuret (copper ions in alkaline
- Specimen should solution ) → Blue/Violet complex
not be hemolyzed
BCP ( Bromcresol - Specific Specimen:
purple) - ↓ in renal - Serum or Heparinized plasma
insufficiency - Prepare 3 tubes
Electrophoresis - For measurement of
(Coomassie Blue) globulin.

-
Computations:

Proteins in Laboratory Non-Protein Nitrogen Containing Compounds

Spectrophotometer Creatinine
- Result of degradation of creatine.
- Can be transformed into ATP
- Excreted by kidneys. With progressive renal
insufficiency there is retention in blood of urea,
creatinine, and uric acid.
- ↑ level may be indicative of renal insufficiency.
- Reference Interval: 0.7-1.4 mg/dL – Male;
0.7-1.2 - Female
- Kinetic Method Principle:
o Creatinine + Sodium picrate → Picrate
Total Protein (Biuret Method) complex (reddish orange)
a. Photometric colorimetric method o Color measured at 490 nm
b. Principle:
o Serum proteins forms a blue/violet
complex when mixed with the biuret
reagents
o Iodide is included as antioxidant
o Color intensity formed is proportional to
the total protein concentration in sample
o Color intensity measures at 540 nm
Reagents

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Urea Uric Acid
- Final result in the metabolism of proteins - Uric and its salts are the end product of the
- Formed in the liver from its destruction purine metabolism.
- ↑ level of urea can be seen in blood in: - ↑ level may be indicative of renal insufficiency
 Diets with excess of proteins and is commonly associated with gout.
 Renal diseases - Reference Interval: 3-7 mg/dL – male;
 Heart failure 2-6 mg/dL- female
 GI hemorrhage - Caraway Method:
 Dehydration o Direct phosphotungstate
 Renal obstruction o Measure at 600 nm
- Reference Interval: 10-50 mg/dL o Uric Acid + buffered phosphate solution
- Berthelot’s Method: + phosphotungstic acid → allantonin +
o Measure at 578 nm Tungsten blue + CO2
o Urea + H2O →urease→2NH3 + CO2
o NH3→salicylate, hypochlorite,
nitroprusside → 2,2-dicarboxyl
indophenol (Blue-green dye)

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