PREPARED BY:

Dayle daniel G. Sorveto, RMT, MSMT

• Comes from the greek word proteis, meaning “ first rank of
importance”
• Are synthesized in the liver and secreted by the hepatocyte into
the circulation except immunoglobulins
• Most important liver function test
• The are macromolecules composed of polymers of covelently
linked amino acids that are involved in every cellular processes
• Proteins are amphoteric
• Effective blood buffers
• They are very effective antigens due to their mlecular mass,
tyrosine content and their specificity
• They provide 12-20% of the total daily body energy requirement
• Proteins are 50%-70% of the cell’s dry weight
1. Repair body tissues
2. Important in blood coagulation and
immunologic function
3. For transport of metabolic
substances
4. Maintenance of osmotic pressure
5. Maintenance of blood pH
6. Biocatalyst
 1. PRIMARY
STRUCTURE
• It determines the identity
of protein, molecular
structure, function
binding capacity and
recognition ability
• Any change in the amino
acid composition can
significantly alter the
protein
2. SECONDARY
STRUCTURE
• It involves the winding of
the polypeptide chain
• It refers to specific 3-
dimensional
conformations-alpha
helix, beta pleated and
bend form
 3. TERTIARY
STRUCTURE
• The folding pattern of the
protein
• Responsible for many of the
physical and chemical
properties of the proteins
• It is maintained by
electrovalent linkages,
hydrogen bonds, disulfide
bridges, Van der waals
forces and Hydrophobic
interactions
4. QUATERNARY
STRUCTURE
• Is the association of 2
or more polypeptide
chain to form an
functional protein
molecule
• Albumin has no
quaternary structures
• E.q: Hgb, LDH, CK
 2. CONJUGATED
1.SIMPLE PROTEINS PROTEINS
• Contain peptide chains • Are composed of a
which on hydrolysis yield protein (apoprotein) and
only amino acids a non protein moiety
• May be fibrous (fbgn, Tn, (prosthetic group)
collagen) or globular (hg, • These proteins impart
plasma CHONS, enzymes, certain characteristics to
peptide hormones) in the proteins
shape
 CONJUGATED PROTEINS
1. Metalloproteins  ferritin, ceruloplasmin,
hgb, and flavoproteins
2. Lipoproteins  VLDL, HDL, LDL,
Chylomicrons
3. Glycoproteins  haptoglobulin, a1-
antitrypsin
4. Mucoproteins or proteoglycans  mucin
(higher CHO content than CHON
5. Nucleoproteins  chromatin (combined
with nucleic acids
“

”
• It migrates ahead of albumin
• It has a half-life of only 2 days
• It is rich in tryptophan and contains 0.5%
carbohydrate
• It has considerable β-pleated sheet
conformation
• It serves as transport protein for T4 and
retinol (Vitamin A)- by complexing with
retinol-binding protein
• It is used as landmark to confirm that the
specimen is really CSF- it crosses more
easily into the CSF that other proteins
• Increased : alcoholism, chronic renal
failure, steroid treatment
• Decreased: poor nutrition
• Reference value: 18-45 mg/dL
• Most abundant protein; Fatty acid
transporter; Calcium and magnesium are
the bound ions
• It is synthesized by the liver
• A general transport protein
• It maintains osmotic pressure
• It is an indicator of nutritional status
• It serves as circulating reservoir of amino
acids
• it is a sensitive and highly prognostic marker
in cases of cystic fibrosis
• It is a “negative Acute Phase Reactant”
• Lowest plasma albumin levels
(abrupt/sudden) are seen in active nephrotic
syndrome (edematous)
• Gradual decrease albumin levels are seen in
total liver damage or cirrhosis
• Reference value: 3.5-5.0 g/dL
• Fastest anodal migration
• it is a group of proteins consist of α1, α2,β,
and γ fractions
• It is usually measured by substracting the
value of serum albumin from the total
protein concentration
• Elevated concentration in early cirrhosis will
balance loss of albumin resulting to normal
levels of total protein
• Measurement: Total Protein – Albumin=
Globulin
• Reference value: 2.3-3.5 g/dL(23-35 g/dL)
 APR? Diagnostic Significance Function
1-Antitrypsin YES Inflammation  Major inhibitor of protease
(AAT) Pregnancy activity- prevents self
Emphysematous destruction
Pulmonary disease  Comprises of 90% of the 1-
Juvenille Hepatic Cirrhosis globulin band

1- Neural tube defect  Post operative marker

Fetoprotein Anencephaly  Detectable in last trimester
(AFP) Spina bifida  Tumor marker:
Fetal distress HEPATIC
Ataxia telang GONODAL cancer
Down Syndrome
Trisomy 18
1-Acid YES Cancer  45% CHO + 11% sialic acid
glycoprotein Pneumonia  Transport progesterone
lorosomucoid Rheumatoid Arthritis  Inactivates lipophilic hormones
Cell Proliferation
1-Anti- YES Infection  Serine proteinase with
chymotrypsin Burn cathepsin G
( 1-X) AMI  Major PSA transporter
Alzheimer’s Disease  1-X is a vital cpt. Of the
Liver Disease amyloid deposits found in
Alzheimer’s disease
 APR? Diagnostic Significance Function
Group Vitamin D transporter
specific-
component
(GC)

Haptoglobin YES Stressful conditions  Prevents the loss of Hb by

Myoglobinuria transporting free hb to the liver
Intravascular hemolysis and its constituent iron into the
urine

Ceruloplasmin YES Infection, Cancer  Copper-binding

Pregnancy  Marker for Wilson’s disease
Wilson’s Disease  Imparts a blue color to protein
Malnutrition  Only protein that has enzymatic
Nephritic disease activity
Menke’s kinky-hair  Vitally important in regulating the
syndome ionic state of iron
2- Nephrotic syndrome  Largest major non-
Macroglobulin Diabetes immunoglobulin
(AMG) Liver disease  Protein in plasma
 Forms a complex with PSA

C-Reactive YES Acute rheumatoid fever  Cardiac marker

Protein (CRP) MI; RA; Gout  Inflammatory marker
Bacterial and Viral
infections
 APR? Diagnostic Function
Significance
β2-Microglobulin Renal Failure  Needed in production of CD8
RA, SLE, HIV  Found on surfaces of nucleated cells
Multiple
myeloma
Common cause
of
Dialysis-associated
Amyloidosis

Transferrin YES Hemochromatosis  Major cpt. Of β2-globulin fraction

(Siderophilin) (-) IDA  Transports ion
APR Liver disease  Prevents loss of Fe++ through kidney
Nephrotic
Syndrome
Malnutrition
Immunoglobulin YES
Lipoprotein
Fibrinogen YES Extensive  Serve as a marker for long-term
coagulation prognosis of cardiovascular disease
Complement YES Inflammation  Participates in immune response
DIC
HA
- Small heme protein found in skeletal and
cardiac muscle
- Transports and stores oxygen
- Higher affinity for oxygen than Hb
- Approximately 2% of the total muscle
protein
- MW: 18 kDa
- Potential nephrotoxin ( it has to be excreted
when plasma concentration exceeds
reference range)
Diagnostic Significance:

 Protein marker to diffuse  Marker for monitoring

out ischemic muscle cell
 Marker for chest pain
(angina) and early  Increased:
detection of Acute
myocardial infarction
(AMI)
*** In AMI screening
test:
Onset: 1-3 hours
Peak level: 5-12 hrs
Normalize: 18-30 hrs
the success or failure
of reperfusion
 AMI
 Angina
 Rhabdomyolysis
 Muscle trauma
 Extrenous exercise
 Intramusculat
injection
 Acute Renal Failure
Methods:

 Measured in SERUM by immunoassay

 Myoglobinuria- it produces a positive dipstick

reaction for occult blood due to
pseudoperoxidase activity

 AMI Value: >100 µg/L

 Complex of 3 proteins ( regulatory proteins) that
binds to the thin filament of cardiac muscle
 Regulators of actin and myosin

 cTnT and cTnI are nearly absent from normal

serum (many healthy individuals have detectable
levels)
 TnC,TnI, TnT = present in both cardiac and skeletal
muscles
*TnC- binds calcium ions that regulate
muscle contractions.
*TnI and TnT- almost absent in normal
serum (AMI Marker)
Reference Value: <0.1
ng/ml (0.1µg/L)
Diagnostic Significance:
 MOST important marker for Cardiac Injury (AMI)

• Cardiac Troponins:
• a. Troponin T (TnT)/ Tropomyosin-binding subunit
• valuable tool in the diagnosis of AMI
• assessment of early and late AMI
• useful in monitoring the effectiveness of thrombolytic
therapy in AMI patients
- elevated in Renal disease and muscular dystrophy
- sensitive marker for the diagnosis of unstable
angina (angina at rest)
 • a. Troponin T (TnT)/ Tropomyosin-binding subunit
• *** In AMI:
• Rises within 3-4 hours after onset of myocardial
damage
• Peak level is at 10-24 hours
• Return to normal in 7 days (but may remain elevated
for (10-14 days)
• Serum levels at or above 1.5ng/ml are considered to be
suggestive of AMI.

b. Troponin I (TnI)/ Inhibitory subunit or Actin-binding unit

- GOLD STANDARD for AMI
- only found in myocardium
- highly specific to AMI (not elevated in renal failure
patients and no detectable amount in the skeletal muscles)
• b. Troponin I (TnI)/ Inhibitory subunit or Actin-
binding unit
- 13 times more abundant in the myocardium than
CK-MB on a weight basis
- Sensitive indicator of Cardiac necrosis
*** In AMI:
• Levels begin to rise 3-6 hours
• Peak in 12-18 hours
• Return to normal in 5-10 days
 Measured in SERUM by immunoassay
Reference values of TnI and TnT as cardiac markers
depend on the antibodies and calibrators used in the
immunoassay
 Cardiac Marker

 Increases in response to peptide (BNP)

ventricular systolic and diastolic dysfunction

 Diagnostic of Congestive heart failure

Specimen: HEPARINIZED Plasma (with or

w/o fasing
 Low molecular protein and a cysteine proteinase inhibitor
 Freely filtered by the glomerulus and completely reabsorbed and
catabolized by the proximal convoluted tubule- produced and
destroyed at a constant rate
 Included in the list of endogenous renal marker owing for its
sensitivity for determining the glomerular filtration rate
 Proposed as an alternate test for serum creatinine and creatinine
clearance test to screen and monitor kidney dysfunction
 Not affected by physiological factors unlike creatinine
 Method: Particle-enhanced immunoturbidimetry,
immunonephelometry
 1. URINARY PROTEINS
o Majority arise from the blood
o Presence of Urine Albumin is generally considered
abnormal
o >20µg/min (normal albumin excretion rate)
o ≥5mg/dL urine protein - color change on urine
dipstick
• PROTEINURIA (>0.5g/day) – result from either
glomerular or tubular dysfunction
 Types of Proteinuria:
a. Glomerular Proteinuria
 Most common and serious type of abnormal proteinuria
 Often called “Albuminuria”

b. Tubular Proteinuria
 Appearance of low molecular mass proteins due to
defective reabsorption

c. Overload Proteinuria
 Includes Hemoglobinuria, Myoglobinuria & Bence-Jones
Proteinuria
d. Postrenal Proteinuria
 Protein from urinary tract caused by infection, bleeding
or malignancy
 MICROALBUMINURIA
• Early indicator of glomerular dysfunction
• Albumin excretion of 30 ug/mg creatinine to 300ug/mg
creatinine (albumin-creatinine ratio)
• 2 out of 3 specimens submitted for testing within three to six-
month period are with abnormal findings.
Increased: Diabetic Nephropathy, fever,infection,
hypertention
Specimen: Random Urine
Method: Random-spot albumin- creatinine ratio
Reference value: 0-29 µg/mg creatinine
Microalbuminuria: 30-300 µg/mg creatinine
Clinical Albuminuria: >300 µg/mg creatinine
 2. CSF PROTEINS
o CSF is an ultrafiltrate of plasma formed in the
choroid plexus of the ventricles of the brain
o CSF glucose and protein analyses- blood sample is
analyzed concurrently
o CSF normally contains very little protein – proteins
in the blood do not cross easily in BBB
o CSF Albumin: 10-30 mg/dL (2/3 of the CSF total
protein
 2. CSF PROTEINS
Method: TCA, SSA,
Coomassie Brilliant Blue Increased: Bacterial,Viral
Dye, Lowry and Kinetic and fungal meningitis,
Biuret Reaction
Traumatic tap, Multiple
sclerosis, Intracerebral
Decreased: Intracranial hemorrhage, Myxedema,
hypertension,
Hyperthyroidism, Leakage
Drug toxicity
of CSF due to trauma

Reference values: 15-45 mg/dL

 1. CSF Oligoclonal banding
 Presence in CSF of 2 or more IgG bands in the
gamma region
 Seen in multiple sclerosis
- 90% of patients show oligoclonal bands in
the gamma region of the protein electrophoresis)
- (+) result fot CSF: ≥ 2 bands in the CSF not
present in serum
 Other disorders with 2 or more bands in the CSF:
 Encephalitis
 Neurosyphilis
 Guillain-Barre Syndrome
 Neoplastic Disorder
 2. Aminoacidopathies
 Inherited disorder of amino acid metabolism
 Exist either the activity of a specific enzyme in the
metabolic pathway or in the membrane transport
system for amino acids
2. a. Alkaptonuria
 Inborn error of metabolism characterized by the
absence of homogentisate oxidase in the tyrosine
pathway
Clinical feature: Ochronosis (tissue pigmentation)
Diagnostic indicator: Darkening of urine upon
standing at RT
 2. b. Homocystinuria
 Characterized by impaired activity of Cystathione-β-
synthetase
 Results to elevated levels of homocysteine and methionine
in blood and urine
Clinical features:
 Physical defects
 Thrombosis
 Osteoporosis
 Eye lens abnormality
 Mental retardation
Screening test:
Modified Guthrie Test (L-methionine
sulfoximine – antagonist)
 2. c. Maple Syrup Urine Disease
(MSUD)
 Characterized by markedly reduced or absence of α-
ketoacid decarboxylase
 Results to accumulation of branched-chain amino acids
(leucine, isoleucine, valine) in blood, urine and CSF)
Clinical features:
 Failure to thrive
 Muscular rigidity
 Mental Retardation
Screening Test: Modified Guthrie Test (4- azaleucine –
antagonist)
Diagnostic Test: Amino Acid Analysis
Indicator: 4mg/dl of Leucine
 2. d. Phenylketonuria
 Autosomal recessive trait characterized by the
deficiency of the enzyme phenylalanine
hydrolase (PAH)/phenylalanine-4-mono-
oxygenase, which catalyzes the conversion of
phenylalanine tyrosine
 Characterized by the presence of phenylpyruvic
acid (prime metabolite) in both blood and urine
in elevated concentration
 Deficiency of tetrahydrobiopterin (BH4)
 Results in elevated blood levels of phenylalanine
 2. d. Phenylketonuria
Reference value for serum phenylalanine: 1.2-3.4
mg/dL(70-200 µmol/L
Clinical features: Retarded mental development
(infants and children)
Diagnostic indicators:
 >1200 µmol/L of phenylalanine in the blood
 “Musty odor” of urine
Screening test: Guthrie Bacterial Inhibition Assay
(Bacillus subtilis spores and β 2-thienylalanine
antagonist)
(+) result = bacterial growth if the phenylalanine
is >4mg/dL
 2. e. Tyrosinemia

 Characterized by the deficiency of either

enzymes:
 Fumarylacetoacetate FAA hydrolase
(Tyrosinemia I)
 Tyrosine aminotransferase (Tyrosinemia II)
 4-hydroxyphenylpyruvic acid oxidase
(Tyrosinemia III)
 Accompanied by elevated methionine and p-
hydroxyphenylpyruvic acid in blood
 Deficiency leads to liver damage or cirrhosis
• It is useful for quantitating the severity of
hepatic dysfunction
• Serum albumin and the Vitamin K-
dependent coagulation factors provide
the most useful indices for assessing severity
of liver disease
• In measuring total proteins in serum, fasting
may not be required
• Analysis of proteins is important for assessing
nutritional status and the presence of severe
diseases involving the liver, kidney and bone
marrow
• Total protein and albumin are about 10%
higher in the ambulatory individuals
• Plasma levels of total protein is 0.2 to 0.4g/dL
higher than serum due to fibrinogen
• Transudates have a total protein of <0.3g/dL
(<50% of the serum total protein); exudates has
>3g/dL
• Is usually performed on serum, which has
no fibrinogen and no anticoagulant that
may slightly dilute proteins in plasma
• Hemolysis may falsely elevate the total
protein
• Reference value: 6.5-8.3 g/dL
• Is the reference method but not routinely
used
• It is based on measurement of the nitrogen
content of the protein
• It uses serum treated with tungstic acid,
forming protein-free filtrate (PFF)
• According to Kjeldahl, 1 gram of nitrogen is
equivalent to 6.54 grams of proteins
• 15.1%-16.8% = nitrogen content of proteins
• Reagent: H2SO4 (digesting agent)
• End product: AMMONIA
• Is the most widely used method,
recommended by the IFCC expert panel
• It is extensively used in clinical labratories,
particularly in automated analyzer in which
protein concentration can be measured
down to 10-15 mg/dL
• It requires at least 2 peptide bonds and an
alkaline medium
• Reference value: 6.5-8.3 g/dL
• Principle: Cupric ions complex the
group involved in the peptide bond
forming a violet-colored chelate
which is proportional to the number of
peptide bonds present and reflects
the total protein level at 540nm.
• Reagents:
• Alkaline Copper Sulfate
• Rochelle Salt (NaK Tartrate)
• NaOH and Potassium Iodide
• It has the highest analytical sensitivity
• Principle: Oxidation of phenolic
compounds such as tyrosine,
tryptophan and histidine to give a
deep blue color
• Main reagent: Phosphotungstic-
molybdic acid or phenol reagent
• Color enhancer: Biuret reagent
• Principle: the absorbance of proteins
at 210nm is due to the absorbance of
the peptide bonds at specific
wavelength
• Proteins absorbs light at 280nm and at
210nm
• Absorption at 280nm is due to
tryphtophan, tyrosine and
phenyalanine
• Principle: migration of charged particles in an
electric field; confirmatory
• The single most significant clinical application
of SPE is for the identification of monoclonal
spike of immunoglobulins and differentiating
them from polyclonal
hypergammglobulinemia, is elevated APR
(AAT, haptoglobin, α1 antichymotrypsin)
• Major proteins that contribute to
electrophoresis: albumin, α1 antitrypsin, a2-
macroglobulin, haptoglobin, β-lpp, transferrin
complement C3, fbgn, & Ig’s
• Confirmatory test for multiple myeloma
 NORMAL SPE PATTERN
ALBUMIN (1ST BAND) – Fastest band
A1-globulin (2nd fastest band)  glycoproteins, AAT (90%), AAG, TBG
A2-globuliin (3rd fastest band)  Ceruloplasmin, Haptoglobin, AMG
Beta-globulin (4th band)  Transferrin (90%), B-lpp, hemopexin, complement (C3
&C4)
Gamma-globulin (5th band; slowest band)  Immunoglobulin and CRP
 REFERENCE VALUES FOR
EACH FRACTION
1. ALBUMIN - 53-65% (3.5-5.0g/dL)
2. A1- globulin - 2.5-5% (0.1-0.3g/dL)
3. A2-globulin - 7-13% (0.6-1.0g/dL)
4. β-globulin - 8-14% (0.7-11g/dL)
5. γ-globulin - 12-22% (0.8-1.6g/dL)
• Is an alternative test to chemical
analysis of serum total proteins
• It is based on measurement of
refractive index of solutes in
serum
• These methods utile sulfosalicylic acid and
or trichloroacetic acid.
• Measurements depends on the formation
of uniform fine precipitate which scatters
incident light in suspension (nephelometry)
or block light (turbidimetry)
• Globulins can be separated from abumin by salting-
out procedures using sodium salts.
• Reagent: Sodium sulfate salt
• The albumin that remains in solution in the
supernatant can be measured by any of the routine
total protein methods; globulin is insoluble in water but
not in dilute salt solution
SOLUBILITY PROPERTY OF PROTEINS
PROTEIN SOLUBLE INSOLUBLE
ALBUMIN Water Saturated salt solution
Concentrated salt Highly concentrated salt solution
solution Hydrocarbon solvents
GLOBULIN Weak salt solution Water
Hydrocarbon solvents Saturated salt solution
Concentrated salt solution
• Coomassie Brilliant Blue Dye is for
detection of proteins to as little ass 1
ug.
• Ninhydrin, which develops a violet
color by reacting with primary amines,
is widely used for detection of
peptides and aa after paper chrom;
aa analyses from ion-exchange
columns; as well ass for detection of
drugs on toxicology screens using thin
layer chrom.
 CLINICAL SIGNIFICANCE
INCREASED TOTAL DECREASED TOTAL
PROTEIN: PROTEIN:

1. Malignancy 1. Hepatic Cirrhosis

2. Multiple Myeloma 2. Glomerulonephritis
3. Waldenstrom’s 3. Nephrotic
macroglobulinemia syndrome
4. Starvation
• It differentiates intrahepatic disorder
(prolonged protime) from extrahepatic
obstructive liver disease (normal protime)
• Prolonged protime despite Vit. K administration
indicate loss of hepatic capacity to synthesize
the proteins
• In acute viral or toxic hepatitis, prolonged
protime signifies massive cellular damage
• Vitamin K is administered inramuscularly, 10mg
daily for 1-3 days
• 2nd Synthetic function test
• Concentration of this protein is inversely
proportional to the severity of the liver disease
• Plasma levels decline when severe
hepatocellular disease last more than 3 weeks
• In hepatic circulatory disorder, albumin is used
because its concentration reflects the shift of
protein and fluid into ascites and its important
contribution to the plasma oncotic pressure
• Decreased serum albumin concentration may
be due to decrease synthesis
• Low total protein + low albumin = hepatic
cirrhosis and nephrotic syndrome
 DYES USED FOR MEASUREMENT:
1. Bromcresol green (BCG)  most commonly used
2. Methyl orange (MO)
3. Hydroxyazobenzene benzoic acid (HABA)
4. Bromcresol Purple (BCP) most specific dye
• Albumin can be measured by direct methods based on its
dye-binding property
• BCG and BCP are cationic dyes, and free from interference
from bilirubin
• BCG and BCP are not significantly affected by the used of
hemolyzed samples
• BCG is used extensively in automatic analyzers for
determining serum albumin in parallel with Biuret reagent for
total protein
• The presence of penicillin may cause falsely low result of
albumin (BCG method)
 ANALBUMINEMIA
1. REDUCED SYNTHESIS  Is hereditary
1. CHRONIC LIVER DSE absence of
1. Severe 2. MALABSORPTION
SNYNDROME
albumin; inability to
synthesize albumin
dehydration 3. MALNUTRITION AND
MUSCLE WASTING
DISEASE BISALBUMINEMIA
2. Prolonged 2. INCREASED LOSS  Is the presence of
tourniquet 1. NEPHROTIC SYNDROME two albumin bands
application- 2.
3.
MASSIVE BURNS
PROTEIN-LOSING
instead of a single
pseudo ENTEROPATHY band in
4. ORTHOSTATIC electrophoresis
hyperalbumin ALBUMNURIA
 It is associated with
emia 3. INCREASED
CATABOLISM excess amount of
1. MASSIVE BURNS therapeutic drugs in
2. WIDESPREAD
MALIGNANCY
serum (penicillin)
3. THYROTOXICOSIS
• It is determined to validate if globulin is higher
than albumin
• If globulin is greater than albumin it is known as
inverted A/G ratio seen in cirrhosis, MM and
Waldenstrom’s macroglobulinemia
• Serum and even urine protein electrophoresis
may help define the clinical situations
• Reference value: 1.3-3.1
PROTEIN LEVELS IN DISEASE STATES

TOTAL PROTEIN ALBUMIN GLOBULIN DISEASE

Cirrhosis, Hepatitis, Obs.

N,
Jaundice

N Nephrosis, Malabsorption

NaCl Retention Syndrome

Dehydration

N Multiple Myeloma