RECENT ADVANCES IN CLINICAL ENZYMOLOGY

TAPESHWAR YADAVP.G Final YearDepartment Of BiochemistryMamata Medical College-Khammam, A.P, INDIA

Measurement of serum enzymes

Diagnostic enzymologyEnzymes are normally intracellular and LOW concentration in bloodEnzyme release (leakage)in the blood indicates cell damage (cell –death, hypoxia, intracellular toxicity)Quantitative measure of cell/tissue damage

Organ specificity- but not absolute specificity inspite of same gene content. Most enzymes are present in most cells-differing amounts

Information from enzymes measurements in serum

Presence of diseaseOrgans involvedAetiology /nature of disease: differential diagnosisExtent of disease-more damaged cells-more leaked enzymes in bloodTime course of disease

SERUM ENZYMES IN HEART DISEASES

1) CK(CPK)2) CK-MB3) Troponin-I/T4) LDH5) AST6) Others

CARDIAC TROPONINS

Not enzymes Now accepted as reliable markers for

myocardial infarction 3 subunits:- 1) Troponin T(TnT): Tropomyosin binding

elements 2) Troponin I(TnI): Actinomyosin ATPase

inhibitory elements 3)Troponin C(TnC): Calcium binding

Cardiac isoform of CTnT & CTnI:- 95% located in myofibrils & 5% cytoplasmic

TnT:- ↑ within 6 hrs of MI & remains ↑ up to 7-14 days. TnI:- released into the blood within 4 hrs after the onset of symptoms of myocardial ischemia; peaks at 14-24 hrs & remains ↑ for 3-5 days post-infarction.

Note:- cardiac troponin ↑ at lower conc. than the 99th percentile value used for MI but still have a risk of having an adverse cardiac event. Troponin-I:- 1 – 10 µg/L

Other Biomarkers

1) H-FABP:- Heart-type fatty acid binding protein Kinetically similar to myoglobin but more

specific to cardiac tissue which contains a greater percentage of this protein than skeletal muscle

May also have role in prediction- prognosis in patients with NSTEMI

Current studies ongoing to further evaluate its utility

2) Brain Natriuretic Peptide(BNP):-

Natriuretic peptide family consists of 3 peptides:- a) atrial natriuretic peptide(ANP) b) brain natriuretic peptide(BNP) c) C-type natriuretic peptide(CNP)

ANP:-produced in cardiac atria.

BNP:- present in human brain but more in cardiac ventricles. pro-BNP- 108A.As High plasma conc. of ANP & BNP :- CHF ↑ BNP- COPD pro-BNP:- best marker of ventricular dysfunction

Myeloperoxidase• MPO is an enzyme that aids white blood cells in

destroying bacteria and viral particles

• MPO catalyzes the conversion of hydrogen peroxide and chloride ions (Cl-) into hypochlorous acid

• Hypochlorous acid is 50 times more potent in microbial killing than hydrogen peroxide

• MPO is released in response to infection and inflammation

• EPIC Norfolk Study showed its predictive value for future cardiovascular disease events in asymptomatic adults.

Summary of MPO

• MPO leads to oxidized LDL cholesterol – Oxidized LDL is phagocytosed by macrophages

producing foam cells*• MPO leads to the consumption of nitric oxide

– Vasoconstriction and endothelial dysfunction• MPO can cause endothelial denuding and

superficial platelet aggregation• MPO indicates activated immune cells

– Activated immune cells and inflammation lead to unstable plaque*

• Inflammatory plaque is inherently less stable– Thin fibrous cap/fissured/denuded

Brennan, NEJM 2003*Hansson, NEJM 2005

CRP

Chronic inflammation also is an important component in the development & progression of atherosclerosis.

Numerous epidemiological studies have demonstrated that ↑ serum CRP conc. are +vely associated with a risk of future CHD events when using an hsCRP assay.

Synthesis- LIVER

CRP:- 0.5 – 1 mg/dl

Clinical Significance:-

Single hs-CRP measurement is a stong predictor of:-

a) MI b) Stroke c) Peripheral vascular disease d) Sudden cardiac death:- individuals without a

history of heart disease.

Direct comparison of traditional and novel biochemical markers of CHD risk, hsCRP is the strongest predictor of future coronary events.

Stefan Blankenberg, MD; Renate Schnabel, MD; Edith Lubos, MD, et al., Myeloperoxidase Early Indicator of Acute Coronary Syndrome and Predictor of Future Cardiovascular Events 2005

ENZYME PROFILE IN LIVER DISEASES

1) ALT2) ALP3) NTP4) GGT

SERUM ENZYMES IN MUSCLE DISEASES

1) CK-MM2) AST3) Aldolase

1.CK-MM

Very much elevated in muscular dystrophies(500-1500 IU/L). Female carriers of X-linked muscular dystrophy (heterozygous)

Raised values:- Secondary muscle disease Hypothyroidism Crush injury Fracture & Cerebrovascular accidents.Actual values depends on:- Severity of the disease & Mass of diseased muscle.

2) AST :- not used now-a-days.

3) Aldolase:- glycolytic enzyme until recent years enzyme of choice more sensitive than ASTSites:- Liver, skeletal muscle & brain. Also found in neoplastic tissues.

Normal Values:- 1.5 – 7 IU/L

Significance of Aldolase:-

Drastically elevated in muscle damages eg- progressive muscular dystrophy poliomyelitis myasthenia gravis multiple sclerosis. Also ↑ in liver diseases, myocardial infarction & Leukemia.

SERUM ENZYMES IN BONE DISEASES

1) Alkaline phosphatase (ALP):- Non-specific enzymes hydrolyses aliphatic, aromatic or heterocyclic

compounds. optimum PH:- betn 9-10. activated by magnesium & manganese. Zn is a constituent ion of ALP.

Produced by osteoblasts of bone associated with calcification process. localised in cell membranes (ecto-enzyme) Also associated with transport mechanisms in liver, kidney & intestinal mucosa.

Normal value:- 40 – 125 IU/L

Iso-enzymes of ALP:-

i) α1 – ALP:- Synthesized by epithelial

cells of biliary canaliculi. about 10% of total activity ↑ in obstructive jaundice.

ii) α2 – ALP:- heat labile Stable at 56o C Produced by hepatic cells About 25% of total ALP

iii) α2 – ALP:- heat stable not be destroyed at 65o C inhibited by Phenylalanine placental origin:- found in blood in normal pregnancy. carcinoplacental iso-enzyme:- carcinoma of lungs, liver & gut. 1% of ALP

iv) pre-β ALP:- heat labile origin- bone 50% of normal ALP ↑ in bone diseases. marker of bone disease

v) γ- ALP:- origin- intestinal cells inhibited by phenylalanine ↑ in ulcerative colitis. 10% of ALP

vi) Leukocyte- ALP:- ↑ in LYMPHOMA↓ in CML

Significance of ALP:-

C) Drastically high levels:- 10-25 times of upper limit bone diseases Osteoblastic activity is enhanced such as paget’s disease, rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bones & hyperparathyroidism.

A) Moderate ↑:- 2-3 times Hepatic diseases such as infective hepatitis, alcoholic hepatitis or hepatocellular carcinoma.

B) Very high:- 10-12 times Extrahepatic obstruction (obs. Jaundice) Intrahepatic cholestasis ( infective hepatitis

ENZYMES IN PROSTATE CANCER

1) Prostate specific antigen (PSA)2) Acid phosphatase (ACP)

1) Prostate specific Antigen(PSA):-

Produced from epithelium of prostate gland. Normally secreted in to seminal fluid. liquefaction of seminal coagulum. serine protease & 32 Kd glycoprotein. Blood- bound to α2-macro-globulin & α1 – antitrypsin.very specific for prostate activity Values above 10µg/L is indicates prostate cancer.

Normal va lue: - 1 – 5 µg /L

2) Acid Phosphatase (ACP):-

an important tumor marker Secreted by prostate cells, RBC, Platelets & WBC.Hydrolyses phosphoric acid ester at PH betn 4-6. ACP of prostate is inhibited by L-tartrate i.e tartrate labile (1 U/L)

Normal values:- 2.5 - 12 IU/L

↑ ed in prostate cancer & highly elevated in bone metastasis of prostate cancer. Marked ↑ in:- ○ Gaucher’s disease, ○ thrombocytosis, ○ chronic granulocytic leukaemia, ○ myeloproliferative disorders etc.Occasional rise in:- ○ paget’s disease, ○ hyperparathyroidism & ○ osteolytic metastasis from breast & other carcinomas.

Total ACP – ACP after tartrate inhibition = prostatic ACP

SERUM ENZYMES IN GI-TRACT DISEASES

1) Serum amylase2) serum lipase

1) SERUM AMYLASE:-

Splits Starch to maltose. activated by Ca2+ & Cl- ions. produced by:- Pancreas & salivary glands.

Normal values:- 50 – 120 IU/L

Isozymes – S1,S2,S3; P1,P2,P3

Clinical significance:- Acute pancreatitis:- Investigation of choice ↑ 1000 times more peak values: 5-12 hrs (after onset of disease) return to normal within 2-4 days.

Moderate ↑:- Chronic pancreatitis. mumps (parotitis) Obstruction of pancreatic duct. Peptic ulcer intestinal obstruction.

Urine amylase:- <375 IU/L ↑ in acute pancreatitis. ↑ on 1st day & remain to be ↑ for 7-10 days.

2) SERUM LIPASE:-

TG ------› MAG + FFA

Present:- pancreatic secretion.

Normal value:- 0.2 - 1.5 IU/L

Significance of lipase:-

Acute pancreatitis:- highly ↑ blood levels. persists for 7-14 days. lipase remains ↑ longer than amylase. not ↑ in mumps.

Moderately ↑:- Carcinoma of pancreas biliary diseases peptic ulcers.

OTHER DIAGNOSTIC ENZYMES

5’-NUCLEOTIDASE

Distribution: liver, bowels, NS, pancreasIncreased in: obstructive type of hepatobiliary diseases Normal values :- 2-15 IU/L

Gamma Glutamyl Transferase (GGT):-

Source: major- Liver & bile ducts. others- pancreas, kidney & prostate gland.

Clinical Significance:- obstructive jaundice Hepatitis Alcoholic cirrhosis

Sensitive indicator:- Alcoholic cirrhosis

NORMAL VALUE:- 10-50 IU/L

Plasma Cholinesterases:-

2 types:- 1) True cholineesterase :- (acetylcholine hydrolase & acetyl-cholineesterase)Present:- nerve tissue & RBC.Function:- hydrolysis of acetylcholine at the synapses & neuromuscular junction.

2) Pseudocholinesterase :- (acetylcholine acyl-hydrolase & Cholinesterase)Present:- liver, heart muscle, intestine & sera.Function:- not known

Clinical Significance:-

Normal value:- 4000 – 12000 IU/L

Liver function test imp. Indicator of insecticide poisoning. ↓ in:- Uremia, shock, anemia, cancer, tuberculosis & malnutrition.

NOTE:-• Degrades succinylcholine, a muscle relaxant given during general anesthesia in surgery.• Some people are deficient in plasma cholinesterase (congenital inherited recessive disease), so the normal dose of succinyl-choline would kill them

Therefore, a determination of plasma cholinesterase is made prior to major surgery.

LEUCINAMINOPEPTIDASE (LAP)

Distribution: liver, kidney, bowels.Increased in: hepatobiliary diseases (obstruction, hepatic metastases).

ORNITHINE CARBAMYL TRASFERASE(OCT)

Ditribution: only liver (hepatospecific).Increased in: liver diseases (hepatitis, cirrhosis, hepatic metastases).

Therapeutic use of enzymesEnzymes Therapeutic applications

1) Asparaginase Acute lymphoblastic leukemia

2) Streptokinase To lyse intravascular clot

3) Urokinase do

4) streptodornase DNAse; applied locally

5) Pancreatin (trypsin & lipase)

Pancreatic insufficiency; oral administration

6) Papain Anti-inflammatory

7) α1 - antitrypsin AAT deficiency; emphysema

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