proteins and clinical enzymology.2012

8/12/2019 Proteins and Clinical Enzymology.2012

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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.

proteins and clinical enzymology.2012

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