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Texto
Proteins and
Clinical
Enzymology
Proteins
Functions of serum proteins
Function Example
Transport Apolipoproteins (cholesterol, triglyceride),
transferrin (iron)
Inflammatory
responses
Acute phase response proteins ( C-
reactive protein, acid glycoprotein )
Humoral immunity Immunoglobulins, complement proteins
Maintenace of
oncotic pressure
All proteins, esp. albumin
Enzymes Renin,coagulation factors, complement
proteins
Blood clotting Coagulation factors, fibrinogen
Protease inhibitors 1-antitrypsin (acts on proteases)
All the plasma proteins are synthesized in liver except gamma globulins.
Composition of principal serum proteins
Class Protein Approximate mean serum
concentration (g/L)
Pre-albumin 0.25
Albumin 40
1-globulin 1-antitrypsin 2.9
1-acid glycoprotein 1.0
2-globulin Haptoglobins 2.0
2-macroglobulin 2.6
Caeruloplasmin 0.35
-globulin Transferrin (Iron) (1) 3.0
Low-density lipoprotein (2) 1.0
Complement proteins (2) 1.0
-globulin IgG 14.0
IgA and others 5.1
Acute phase proteins
It is a complex range of physiological changes that occur
following infection, inflammation, trauma, burns and other
related conditions.
Changes in a wide range of proteins which would act to
restore homeostasis to the body and is mediated by cytokines.
Examples of acute phase proteins are:
Positive acute phase proteins C-Reactive Protein (CRP),
caeruloplasmin, haptoglobin, 2-macroglobulin, 1-antitrypsin,
1-acid glycoprotein, Mannan-binding lectin, fibrinogen
(whose concentration increases during APR).
Negative acute phase proteins Albumin, prealbumin,
transferrin (whose concentration decreases during APR).
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The production of these proteins is stimulated by pro-
inflammatory cytokines (Interleukin-1, Tumour necrosis alpha
and Interleukin-6) released by macrophages.
Different response to inflammation.
C-Reactive protein (CRP) bind to macromolecules
released by damaged tissue or infective agents and
promote their phagocytosis interactions with
humoral and cellular effectors of inflammation.
Caeruloplasmin Major transport protein for copper
Haptoglobin Binds free haemoglobin
1-antitrypsin Protease inhibitor
1-acid glycoprotein Inhibits platelet aggregation
Transferrin Iron transport
C-Reactive Protein (CRP)
CRP is an inflammatory marker. Used for monitoring
patients with inflammatory conditions such as rheumatoid
arthritis and artherosclerosis.
More sensitive and specific measurement than
erythrocytes sedimentation rate (ESR).
Its concentrations begin to rise at about 8hrs after APR
and reaches a peak after 48hrs before falling.
Prospective studies have shown that an elevated level of
CRP is associated with increased risk for cardiovascular
events in apparently healthy personsalso used as a
marker for CHD
Serum protein electrophoresis is a laboratory test that examines
specific proteins in the blood.
Electrophoresis is a method of separating proteins based on their
physical properties (i.e. size, charge, shape).
Serum is placed on special paper (cellulose acetate) treated with
agarose gel and exposed to an electric current to separate the
serum protein components.
The net charge (positive or negative) and the size and shape of
the protein commonly are used in differentiating various serum
proteins.
The pattern of serum protein electrophoresis results depends on
the fractions of two major types of protein: albumin and globulins.
Serum protein electrophoresis
Albumin, the major protein component of serum, is produced
by the liver under normal physiologic conditions.
Globulins comprise a much smaller fraction of the total
serum protein content. The subsets of globulins are 1-
globulins, 2-globulins, -globulins, and -globulins.
Positive
electrodes
Negative
electrodes
Normal typical pattern of protein electrophoresis
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Interpretation of
serum
electrophoresis
results
Serum protein electrophoresis results
during acute phase response
Plasma protein levels display reasonably predictable
changes in response to acute inflammation, malignancy,
trauma, necrosis, infarction, burns, and chemical injury
an acute phase protein pattern.
An increase in positive APP, a decrease in negative APP
Serum protein electrophoresis
Serum protein electrophoresis is commonly performed when
multiple myeloma is suspected.
In the interpretation of serum protein electrophoresis, most
attention focuses on the gamma region, which is composed
predominantly of antibodies of the IgG type.
Diseases that produce an increase in the gamma-globulin level
include malignant lymphoma, chronic lymphocytic leukemia,
liver diseases and multiple myeloma.
Abnormal serum
protein
electrophoresis
pattern in a patient
with multiple
myeloma.
** Note the large
spike in the
gamma region.
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Multiple Myeloma (Myelomatosis)
Caused by malignant proliferation of plasma
cells throughout the bone marrow.
Conditions increasing frequent after the age
of 50, even though can occur at 30, but rare.
Disordered immunoglobulin synthesis and/or
secretion from the cell.
Symptoms?
SPE shows paraproteinaemia shows
appearance of an abnormal, narro to dense
band commonly in the -region on the
electrophoretic strip.
Urinary protein electrophoresis also shows
the presence of Bence-Jones protein (BJP)
band in urine (Picture: patient B=heavy BJP
and leakage of albumin and other low
molecular weight proteins (glomerular
permeability) in urine).
Multiple myeloma:
Approx 80% of pts have
chief complaint of bone
pain w/ diffuse bone
tenderness.
It develops as multiple
painful lesions (punched-
out areas) throughout
skeleton, especially the
skull, ribs, vertebrate and
pelvis; generalised
osteoporosis: normal
alkaline phosphatase
activity, anaemia, renal
failure, infection
Bone Marrow Aspirate with
malignant plasma cells
Much rouleaux formation of
the red cells
Serum/Plasma Enzymes
Measurements of the activity of enzymes in serum/plasma
are of value in the diagnosis and management of a
wide range of disease (especially for diagnosis of
inherited metabolic diseases). Enzyme activity in
plasma is expressed in U/L.
Small amounts present in the blood as a result of normal
cell turnover.
Increased amounts could indicate:
1) Tissue damage
2) Increased cell turnover
3) Cellular proliferation (i.e. neoplasia)
4) Increased enzyme synthesis
5) Obstruction to secretion
6) Decreased clearance
Disadvantages of enzymes assays
1) Lack of specificity to a particular tissue or cell
type
2) Time of sample taken
Examples of enzymes
1) Alkaline phosphatase (ALP),
2) Aminotransferase (ALT/AST),
3) Gamma-Glutamyl Transferase,
4) Lactate dehydrogenase (LDH),
5) Creatine kinase (CK),
6) Amylase
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Lactate dehydrogenase (LDH)
In medicine, LDH is often used as a marker of tissue
breakdown (i.e. in cancer cases). As LDH is abundant
in red blood cells and can function as a marker for
hemolysis.
It can also be used as a marker of myocardial
infarction. Following a myocardial infarction, levels of
LDH peak at 3-4 days and remain elevated for up to 10
days.
Increases in plasma LDH activity indicates acute
damage to the liver (main), kidneys, skeletal muscle,
haemolytic anaemia and MI.
There are 5 isoenzymes of LDH.
LDH-1 to LDH-5.
LDH -1 is found predominantly in cardiac tissue (and
also erythrocytes) while LDH-5 is mainly found in the
skeletal muscle and liver.
Isoenzyme profiling following electrophoresis:
Densitometric patterns of LDH isoenzymes
Creatine kinase (CK)
CK is an enzyme found mainly in the heart, brain,
and skeletal muscle.
When the total CK level is very high, it usually means
there has been injury or stress to the heart, the brain,
or muscle tissue.
Measurement of CK have long being used to assist
in the diagnosis of myocardial infarction.
The enzymatic active CK molecule is in dimer form;
there are 2 monomers, M and B. So there are 3
isoenzymes:
BB (confined to brain),
MM (mainly in blood plasma and skeletal muscle) &
MB (in cardiac muscle (30%)).
The first enzyme to increase is creatine kinase MB
(CK-MB), followed by total CK, AST and lactate
degydrogenase (LDH).
LDH (so which
isoenzyme??)
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Tumour Markers
Tumour markers are substances, usually proteins, that are
produced by tumour cells or by other cells of the body in
response to cancer or certain benign (noncancerous)
conditions.
Some tumour markers are specific, while others are seen in
several cancer types.
Some people are at a higher risk for particular cancers
because they have inherited a genetic mutation. Example:
BRCA1 and BRCA2 are genetic testing to investigate the
inherited risk of breast cancer and ovarian cancer.
Tumor markers can be used for one of five purposes:
Ideal tumour
marker
Screening
Diagnosis
Prognosis Monitoring
Follow up to detect
recurrence
Characteristics of an ideal tumour markers
1. Specificity for a single type of cancer
2. High sensitivity for cancerous growths
3. Correlation of marker level with tumour size
4. Short half-life in circulation
Types of tumour markers
Enzymes (i.e. acid phosphatase)
Hormones (i.e. calcitonin, ACTH, hCG)
Fetal antigen (i.e. AFP, CEA)
Carcinoma-associated antigens (CA) (i.e. CA 125, CA 15-3,
CA 19-9)
Proteins (i.e. IgM, immunoglobulins, Bence Jones proteins)
Genetic mutations (BRCA1/2, p53, c-myc)
Some clinically useful markers
MARKER TUMOUR USES
-fetoprotein (AFP) Hepatocarcinoma
Germ cell tumour (teratoma)
SDMF
DPMF
-human chorionic gonadotrophin
(HCG)
Germ cell tumour
Choriocarcinoma
DPMF
SDPMF
Carcinoembryonic antigen (CEA) Colorectal carcinoma MF
Paraproteins Multiple myeloma DMF
Prostate specific antigen (PSA) Prostate carcinoma MF
Acid phosphatase Prostate carcinoma MF
CA125 Ovarian cancer SPM
CA15.3 Breast cancer SDMF
CA19.9 Pancreatic, colorectal
cancer
SDMF
S-Screening; D-Diagnosis; P-Prognosis; M-Monitoring treatment, F-Follow-up
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Combined markers
Different types of tumours at different stages of development
produce a variety of different tumour markers at various
concentrationstherefore, lack sensitivity and specificity
required to identify a specific type of cancer.
Quantitative assays for multiple tumour markers are
becoming increasingly valuable tool in clinical and differential
diagnosis of cancer.
MARKERS MAJOR CLINICAL APPLICATION
hCG and AFP Staging and monitoring testicular cancer
CEA and CA 19-9 Pancreatic cancer, colorectal cancer
CEA and CA 15-3 Breast cancer
CA 125 and CA 15-3 Ovarian cancer
CA 125/CEA ratio Differentiate ovarian from colon cancer
Genetic markers
In the progression of normal cells to cancer, various
changes occur to genes involved in cell division.
Targets for these genetic changes:
1) Proto-oncogenes are normal genes that code
mainly for proteins that control normal cell growth
(point mutationdefective oncogenes and
overexpressed oncoproteins)
2) Tumour suppressor genes are normal genes
found in cells that prevent or inhibit cancerous
processes (deletion/mutational inactivationincrease
the probability of formation of tumour)
MARKERS FUNCTION MAJOR CANCER
Mutations in Oncogenes
c-myc Transcription regulation Lymphoma
N-ras Signal transduction Leukemia, neuroblastoma
K-ras Signal transduction Predictive markers for colorectal and
K-ras variant ovarian cancer
HER2/neu Signal transduction Predictive markers for higher
aggressiveness in breast cancers
Mutations in Tumour Supppessor Genes
p53 Control cell division Breast, colon, lung, brain, leukemia
BRCA1 & Cell cycle & division Breast and ovarian
BRCA2
Genetic tumour markers
In general, tumour markers cannot be used alone to
diagnose cancer; they must be combined with other tests
such as a biopsy, to diagnose cancer.
Tumour marker levels may be measured before treatment
to help doctors plan appropriate therapy.
In some types of cancer, tumour marker levels reflect the
stage (extent) of the disease.
Tumour marker levels also may be used to check how a
patient is responding to treatment.
A decrease or return to a normal level may indicate that the cancer
is responding to therapy, whereas an increase may indicate that
the cancer is not responding.
After treatment has ended, tumour marker levels may be used to
check for recurrence (cancer that has returned).
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Examples of tumour
markers used in clinical
practice and cancer
In Malaysia, one in 20 women is at risk of developing breast
cancer and about 30% of Malaysian women are now suffering
from it.
CA153 and Breast Cancer
Breast cancer was the most common overall
cancer and the most common cancer in
women amongst all races from the age of 20
years old.
The incidence was highest among the
Chinese followed by the Indians and then,
the Malays.
The cumulative life time risk of developing
breast cancer for Chinese women, Indian
women and Malay women were 1 in 16, 1 in
17 and 1 in 28 respectively.
The peak incidence appeared to be 50-59
years old in Malaysia.
Breast cancer may be invasive or noninvasive.
Invasive means it has spread from the milk
duct or lobule to other tissues in the breast.
Noninvasive means it has not yet invaded
other breast tissue. Noninvasive breast cancer
is called "in situ.
ER-positive cancer
Many breast cancers are sensitive to the hormone oestrogen. Oestrogen causes
the breast cancer tumour to grow. Such cancers have oestrogen receptors on
the surface of their cells. They are called oestrogen receptor-positive cancer or
ER-positive cancer. Hormonal therapy is used.
HER2-positive breast cancer-
HER2 (Human epidermal growth factor receptor, or HER2/neu)
refers to a gene that helps cells grow, divide, and repair themselves. When cells
(including cancer cells) have too many copies of this gene, they grow faster.
Historically, women with HER2-positive breast cancer have a more aggressive
disease and a higher risk that the disease will return (recur) than women who do
not have this type. Targeted treatments against HER2 is used. One such drug is
trastuzumab (Herceptin).
CA 15-3 is not sensitive or specific enough to be considered useful as a
tool for cancer screening.
Its main use is to monitor a person's response to breast cancer treatment
and to watch for breast cancer recurrence.
CA 15-3 may be ordered along with other tests, such as oestrogen and
progesterone receptors, Her2/neu for breast cancer, when advanced
breast cancer is first diagnosed to help determine cancer characteristics
and treatment options.
In general, the higher the CA 15-3 level, the more advanced the breast
cancer and the larger the tumour burden. CA 15-3 concentrations tend to
increase as the cancer grows.
In metastatic breast cancer, the highest levels of CA 15-3 often are seen
when the cancer has spread to the bones and/or the liver.
CA 15-3 can be elevated in malignancies other than breast cancer,
including lung, pancreatic, ovarian, liver, and colorectal cancers.
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Cancer of the colon and rectum.
Together, cancers of the colon and
rectum are 2
nd
most common cancers in
Malaysia, after breast cancer. About
3600 new cases are diagnosed every
year.
They occur in both men and women and
are most often found among people
who are over the age of 50.
Colorectal cancer is the most common
form of cancer in males in Malaysia.
CEA and Colorectal cancer
CEA
Described by Gold and Freedman in 1965 as a marker for
Colorectal Cancer
Molecular mass of approximately 200,000 kDA. Glycoprotein
with a carbohydrate composition ranging from 50 - 85% of
molecular mass
CEA levels 5 - 10 times upper limit of normal suggests colon
cancer
CEA is not used to screen for colon cancer, but to monitor
effectiveness of, or recurrence after, treatment
PSA and Prostate Cancer
Prostate cancer was the second most common cancer in Indian
males. The cancer ranked fourth among male cancers in
Malaysia (MAKNA, 2012). It was highest among the Indians
(13.7 per 100,000) followed by Chinese (11.5 per 100,000) and
Malay (9.2 per 100,000).
In Asia, prostate cancer has become one of the leading male
cancers with the incidence having risen rapidly in the last 20
years.
Affects 1:1470 males in Malaysia.
PSA
A marker for prostatic carcinoma, a common male tumour.
A 33kDa glycoprotein manufactured almost exclusively by
the prostate gland, with a half-life of about 3 days.
PSA is normally present in the blood at very low levels.
The reference range of 0-4.0 ng/mL.
Levels raised in benign prostatic hyperplasia (BPH) and
prostatic carcinoma. Also raised in prostate infection, and
after rectal examination and recent ejaculation.
Plasma PSA greater than 10ng/mL are strongly suggestive
of carcinoma. Greater than 20ng/mL is suggestive of PC
that has spread beyong the prostate gland.
Levels also increase with age.
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Limitations: overlap between BPH and prostatic carcinoma
PSA is bound in the plasma to either 1-antichymotripsin or
2-macroglobulin.
Levels of bound PSA (1-antichymotripsin) is higher in PC,
whereas free PSA is higher in BPH
Ratio free: total PSA (%) > 17% BPH;
< 17% PC
PSA
Cerebrospinal fluid (CSF)
Cerebrospinal fluid (CSF) is a clear bodily fluid
that occupies the subarachnoid space in the
brain (the space between the skull and the
cerebral cortex) and also the spinal cord.
It is a very pure saline solution with microglia
and acts as a "cushion" or buffer for the cortex.
Cerebrospinal fluid (CSF): Diagnosis
Cerebrospinal fluid can be tested for the diagnosis of a
variety of neurological diseases.
It is usually obtained by a procedure called lumbar
puncture.
Common tests performed on CSF include protein and
glucose levels, cell counts and differential, microscopic
examination, and culture.
These parameters alone may be extremely beneficial in
the diagnosis of subarachnoid hemorrhage and central
nervous system infections (such as meningitis).
Moreover, a cerebrospinal fluid culture examination may
yield the microorganism that has caused the infection.
Lumbar puncture
Lumbar puncture
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Meningitis is the inflammation of the protective membranes
covering the central nervous system.
Meningitis is a serious condition owing to the proximity of
the location to the brain and spinal cord. The potential for
serious damage to motor control, thought processes, or
even death
Needs urgent medical attention.
CSF fluid stained with India
ink showing Cryptococcus
neoformans
Most cases of meningitis are caused by
microorganisms, such as viruses,
bacteria, fungi, or parasites, that spread
into the blood and into the cerebrospinal
fluid (CSF).
Cerebrospinal fluid (CSF): Meningitis
Rapid diagnostic test: Meningitis
Provides a
positive result if
there is a
detectable level
of antigen in the
sample.