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Proteins andClinicalEnzymology
Proteins
Functions of serum proteins
Function Example
Transport Apolipoproteins (cholesterol, triglyceride),transferrin (iron)
Inflammatoryresponses
Acute phase response proteins ( C-reactive protein, acid glycoprotein )
Humoral immunity Immunoglobulins, complement proteins
Maintenace ofoncotic pressure
All proteins, esp. albumin
Enzymes Renin,coagulation factors, complementproteins
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 1.02-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 occurfollowing infection, inflammation, trauma, burns and otherrelated conditions.
Changes in a wide range of proteins which would act torestore 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 alphaand Interleukin-6) released by macrophages.
Different response to inflammation.
C-Reactive protein (CRP) bind to macromoleculesreleased by damaged tissue or infective agents andpromote their phagocytosis interactions withhumoral 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 monitoringpatients with inflammatory conditions such as rheumatoidarthritis and artherosclerosis.
More sensitive and specific measurement thanerythrocytes sedimentation rate (ESR).
Its concentrations begin to rise at about 8hrs after APRand reaches a peak after 48hrs before falling.
Prospective studies have shown that an elevated level ofCRP is associated with increased risk for cardiovascularevents in apparently healthy persons also used as amarker 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 theirphysical properties (i.e. size, charge, shape).
Serum is placed on special paper (cellulose acetate) treated withagarose gel and exposed to an electric current to separate theserum protein components.
The net charge (positive or negative) and the size and shape of
the protein commonly are used in differentiating various serumproteins.
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 producedby the liver under normal physiologic conditions.
Globulins comprise a much smaller fraction of the totalserum protein content. The subsets of globulins are 1-globulins, 2-globulins, -globulins, and -globulins.
Positiveelectrodes
Negativeelectrodes
Normal typical pattern of protein electrophoresis
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Interpretation of
serumelectrophoresis
results
Serum protein electrophoresis resultsduring acute phase response
Plasma protein levels display reasonably predictablechanges in response to acute inflammation, malignancy,trauma, necrosis, infarction, burns, and chemical injuryan acute phase protein pattern.
An increase in positive APP, a decrease in negative APP
Serum protein electrophoresisSerum protein electrophoresis is commonly performed whenmultiple myeloma is suspected.
In the interpretation of serum protein electrophoresis, mostattention focuses on the gamma region, which is composedpredominantly of antibodies of the IgG type.
Diseases that produce an increase in the gamma-globulin levelinclude malignant lymphoma, chronic lymphocytic leukemia,liver diseases and multiple myeloma.
Abnormal serumprotein
electrophoresispattern in a patientwith multiple
myeloma.** Note the largespike 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 ageof 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 denseband commonly in the -region on theelectrophoretic strip.
Urinary protein electrophoresis also shows
the presence of Bence-Jones protein (BJP)band in urine (Picture: patient B=heavy BJPand leakage of albumin and other low
molecular weight proteins (glomerularpermeability) in urine).
Multiple myeloma:Approx 80% of pts havechief complaint of bonepain w/ dif fuse bonetenderness.It develops as multiplepainful lesions (punched-
out areas) throughoutskeleton, especially theskull, ribs, vertebrate andpelvis; generalised
osteoporosis: normalalkaline phosphataseactivity, anaemia, renalfailure, infection
Bone Marrow Aspirate withmalignant plasma cells
Much rouleaux formation ofthe red cells
Serum/Plasma Enzymes
Measurements of the activity of enzymes in serum/plasma
are of value in the diagnosis and management of awide range of disease (especially for diagnosis ofinherited metabolic diseases). Enzyme activity inplasma is expressed in U/L.
Small amounts present in the blood as a result of normalcell 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 enzymes1) 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 abundantin red blood cells and can function as a marker forhemolysis.
It can also be used as a marker of myocardial
infarction. Following a myocardial infarction, levels ofLDH peak at 3-4 days and remain elevated for up to 10days.
Increases in plasma LDH activity indicates acutedamage 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 (andalso erythrocytes) while LDH-5 is mainly found in theskeletal 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 meansthere has been injury or stress to the heart, the brain,or muscle tissue.
Measurement of CK have long being used to assistin the diagnosis of myocardial infarction.
The enzymatic active CK molecule is in dimer form;there are 2 monomers, M and B. So there are 3isoenzymes:
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 lactatedegydrogenase (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 inresponse to cancer or certain benign (noncancerous)conditions.
Some tumour markers are specific, while others are seen inseveral cancer types.
Some people are at a higher risk for particular cancersbecause 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 tumourmarker
Screening Diagnosis
PrognosisMonitoring
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, colorectalcancer
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 variousconcentrations therefore, lack sensitivity and specificityrequired to identify a specific type of cancer.
Quantitative assays for multiple tumour markers arebecoming increasingly valuable tool in clinical and differentialdiagnosis 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, variouschanges occur to genes involved in cell division.
Targets for these genetic changes:
1) Proto-oncogenes are normal genes that codemainly for proteins that control normal cell growth
(point mutation defective oncogenes andoverexpressed oncoproteins)
2) Tumour suppressor genes are normal genesfound in cells that prevent or inhibit cancerousprocesses (deletion/mutational inactivation increasethe 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 a ndK-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 ovarianBRCA2
Genetic tumour markers In general, tumour markers cannot be used alone to
diagnose cancer; they must be combined with other testssuch as a biopsy, to diagnose cancer.
Tumour marker levels may be measured before treatmentto help doctors plan appropriate therapy.
In some types of cancer, tumour marker levels reflect thestage (extent) of the disease.
Tumour marker levels also may be used to check how apatient 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 thatthe cancer is not responding.
After treatment has ended, tumour marker levels may be used tocheck for recurrence (cancer that has returned).
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Cancer of the colon and rectum.
Together, cancers of the colon andrectum are 2nd most common cancers inMalaysia, after breast cancer. About3600 new cases are diagnosed everyyear.
They occur in both men and women andare most often found among peoplewho are over the age of 50.
Colorectal cancer is the most commonform of cancer in males in Malaysia.
CEA and Colorectal cancerCEA
Described by Gold and Freedman in 1965 as a marker forColorectal Cancer
Molecular mass of approximately 200,000 kDA. Glycoproteinwith a carbohydrate composition ranging from 50 - 85% ofmolecular mass
CEA levels 5 - 10 times upper limit of normal suggests coloncancer
CEA is not used to screen for colon cancer, but to monitoreffectiveness of, or recurrence after, treatment
PSA and Prostate CancerProstate cancer was the second most common cancer in Indian
males. The cancer ranked fourth among male cancers inMalaysia (MAKNA, 2012). It was highest among the Indians(13.7 per 100,000) followed by Chinese (11.5 per 100,000) andMalay (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 20years.
Affects 1:1470 males in Malaysia.
PSA
A marker for prostatic carcinoma, a common male tumour.
A 33kDa glycoprotein manufactured almost exclusively bythe 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) andprostatic carcinoma. Also raised in prostate infection, andafter rectal examination and recent ejaculation.
Plasma PSA greater than 10ng/mL are strongly suggestiveof 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 or2-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 thebrain (the space between the skull and thecerebral cortex) and also the spinal cord.
It is a very pure saline solution with microgliaand 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 lumbarpuncture.
Common tests performed on CSF include protein andglucose levels, cell counts and differential, microscopicexamination, and culture.
These parameters alone may be extremely beneficial inthe diagnosis of subarachnoid hemorrhage and centralnervous system infections (such as meningitis).
Moreover, a cerebrospinal fluid culture examination mayyield the microorganism that has caused the infection.
Lumbar puncture
Lumbar puncture
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Meningitis is the inflammation of the protective membranescovering the central nervous system.
Meningitis is a serious condition owing to the proximity of
the location to the brain and spinal cord. The potential forserious damage to motor control, thought processes, oreven death
Needs urgent medical attention.
CSF fluid stained with Indiaink showing Cryptococcus
neoformans
Most cases of meningitis are caused by
microorganisms, such as viruses,bacteria, fungi, or parasites, that spreadinto the blood and into the cerebrospinalfluid (CSF).
Cerebrospinal fluid (CSF): Meningitis Rapid diagnostic test: Meningitis
Provides apositive result ifthere is adetectable levelof antigen in thesample.