ENZYMOLOGY(SPECIFIC ENZYMES)

Rolaine Ann Anoza

CARDIAC MARKERS

1. CREATININE KINASE (CK)

2. LACTATE DEHYDROGENASE (LDH)

3. ASPARTATE AMINOTRANSFERASE (AST) OR SERUM GLUTAMIC OXALOACETATE TRANSAMINASE (SGOT)

4. MYOGLOBIN

5. CARDIAC TROPONINS

CREATININE KINASE (ATP:creatinine N-phosphotransferase, EC 2.7.3.2)***primary clinical use is detection of AMI

3 ISOENZYMES:

CK1CK1 most anodalmost anodal (BB)(BB)CK2 CK2 (MB)(MB)CK3 most cathodalCK3 most cathodal (MM) (MM)

TISSUE DISTRIBUTION:• in myocardium - 80% CK-MM80% CK-MM

20% CK-MB• skeletal muscle- >99% CK-MM>99% CK-MM

<1% CK-MBCOMBINED ISOENZYME ANALYSIS RULE OUT MYOCARDIAL COMBINED ISOENZYME ANALYSIS RULE OUT MYOCARDIAL

INFARCTION (MI)INFARCTION (MI)

CK-MB AbsentCK-MB Present &

Usual LDCK-MB Present &

Flipped-LD

100% predictivevalue that

there is no MI

Both MI and non-MIcases

100% predictivevalue that

there is MI

CK IN ACUTE MYOCARDIAL INFARCTION (AMI):

CK IN ACUTE MYOCARDIAL INFARCTION (AMI):

INCREASES: 4-6 hours after the onset

PEAKS: 12 hours post-AMI

RETURNS TO NORMAL: 24 hours after

INCREASES: 4-6 hours after the onset

PEAKS: 12 hours post-AMI

RETURNS TO NORMAL: 24 hours after

Pronounced elevation (5 or more timesPronounced elevation (5 or more times Normal)Normal)– Duchenne’s muscular dystrophy– Polymyositis (skeletal muscles inflammation)– Dermatomyositis (skin inflammation)– AMI

Mild or moderate elevation (2-4 times Normal)Mild or moderate elevation (2-4 times Normal)– severe exercise, trauma, surgical procedure, IM

injections – delirium tremens, alcoholic myopathy– MI, severe ischemic injury– pulmonary infarction– pulmonary edema– hypothyroidism– acute agitated psychoses

LACTATE DEHYDROGENASE (LD; L-lactate:NAD oxidoreductase, EC 1.1.1.27)

Reversible conversion of lactate to pyruvate using conversion of lactate to pyruvate using NADNAD++ as cofactor as cofactor

NORMAL LDH1 TO LDH2 RATIO:Normal LDH1:LDH2 = 0.5

*LDH1<LDH2

In AMI:• LDH 1 is increased, there is LDH FLIPPED RATIO

(LDH1 > LDH2)

Normal LDH1:LDH2 = 0.5

*LDH1<LDH2

In AMI:• LDH 1 is increased, there is LDH FLIPPED RATIO

(LDH1 > LDH2)

Isoenzyme Isoenzyme chain chain

CompositionComposition ~% normally ~% normally present in present in serum serum

Tissue rich in Tissue rich in the the isoenzymeisoenzyme

LD1 (most anodal)

HHHH 29-37 heart, brain, RBC

LD2 HHHM 42-48 heart, brain, RBC

LD3 HHMM 16-20 brain, kidney, lung

LD4 HMMM 2-4 Liver, skeletal muscle, kidney

LD5 (most cathodal)

MMMM 0.5-1.5 Liver, skeletal muscle, kidney

LD IN ACUTE MYOCARDIAL INFARCTION (AMI):

INCREASES: 24 hours after the onset

PEAKS: 48 hours post-AMI

RETURNS TO NORMAL: 12 days after

INCREASES: 24 hours after the onset

PEAKS: 48 hours post-AMI

RETURNS TO NORMAL: 12 days after

CONDITIONS AFFECTING TOTAL LD ACTIVITYCONDITIONS AFFECTING TOTAL LD ACTIVITY

Pronounced Elevation (5 or more times Normal)Pronounced Elevation (5 or more times Normal) Megaloblastic anemiaMegaloblastic anemia Widespread carcinomatosis, esp. hepatic metastasesWidespread carcinomatosis, esp. hepatic metastases Septic shock and hypoxiaSeptic shock and hypoxia HepatitisHepatitis Renal infarctionRenal infarction Thrombotic thrombocytompenic purpuraThrombotic thrombocytompenic purpura

Moderate Elevation (3-5 times Normal)Moderate Elevation (3-5 times Normal) MIMI Pulmonary infarctionPulmonary infarction Hemolytic conditionsHemolytic conditions LeukemiaLeukemia IMIM Delirium tremensDelirium tremens Muscular dystrophyMuscular dystrophy

MYOGLOBIN• Small muscular protein

• storage & transfer of O2

• has higher affinity for O2 than Hb (favoring muscle stores of O2)

• first protein marker to diffuse out of ischemic muscle cellsfirst protein marker to diffuse out of ischemic muscle cells• effective for RULING OUT AMI when its conc. remain low• presumptive for AMI (not specific, only sensitive)presumptive for AMI (not specific, only sensitive)• Peaks 6 hours post-AMI and returns to normal after 24 Peaks 6 hours post-AMI and returns to normal after 24

hourshours• stable in whole blood or serum kept refrigerated for several

days

TROPONINS• Mediators of muscle contraction

• consists of 3 polypeptides (troponic complex)

troponin : CC- smallest; binds Ca++

II- inhibitory; binds action

TT- largest; binds tropomyosin

• I & T released into circulation following myocardial I & T released into circulation following myocardial injury (highly specific)injury (highly specific)

TROPONIN I - CARDIO-SPECIFIC

TROPONIN T- MOST SENSITIVE BUT NOT SPECIFIC

***TROPONINS INCREASE 4-8 HOURS, PEAK 24 HOURS AFTER AND RETURN TO NORMAL 5-10 DAYS POST-AMI.

PHOSPHATASES- Act on compounds with single POsingle PO44 groups groups

1. ALKALINE PHOSPHATASE (ALP) OR ALKALINE ORTHOPHOSPHORIC

MONOESTER PHOSPHOHYDROLASE

2. ACID PHOSPHATASE (ACP) OR ACID ORTHHOPHOSPHORIC

MONOESTEER PHOSPHOHYDROLASE

ALP (EC 3.1.3.1, optimum pH10)•ALP- widely distributed along the surface membranes of metabolically active cells•Marker for obstructive jaundice, hepatic and bone disorders*high in bone, liver, intestine, kidney, WBCs *high in bone, liver, intestine, kidney, WBCs & placenta& placentaheat fractionation-heat fractionation- most common method of most common method of distinguishing ALP isoenzymesdistinguishing ALP isoenzymes (serum heated at 56(serum heated at 56OOC for 15 min. & C for 15 min. & assayed for remaining ALP activity)assayed for remaining ALP activity)

*Fasting blood samples- red top; EDTA chelates Zn++ in ALP so unsatisfactory

*sensitive at low temperature (4C), leads to increased value

*HIGHEST ELEVATION IN PAGET’S DISEASE

ISOENZYMES OF ALP:

1.LIVER (HEAT STABLE)2.BONE (HEAT LABILE)3.INTESTINE4.PLACENTA5.KIDNEY6.REGAN (CARCINOMA)* Up to 16 isoenzymes

TO DISTINGUISH THE INCREASE OF ALP (BONE OR LIVER), USE GGT ENZYME:

LIVER= INC. ALP + GGTBONE = INC. ALP + NORMAL

GGT

METHODS OF ALP MEASUREMENT:

METHOD SUBSTRATE END PRODUCTS

1. SHINOWARA BETA-GLYCEROPO4 INORGANIC PO4 + GLYCEROL

2. KING AND ARMSTRONG

PHENYLPHOSPHATE PHENOL

3. BESSY LOWRY AND BROCK (COMMONLY USED)

PARA-NITROPHENYL PO4

PARA-NITROPHENOL OR YELLOW NITROPHENOXIDE ION

4. HUGGINS AND TALALAY

PHENOLPHTHALEIN DI-PO4

PHENOLPHTHALEIN RED

5. MOSS ALPHA-NAPHTHOL PO4 ALPHA-NAPHTHOL

6. KLEIN, BABSON AND READ

BUFFERED PHENOLPHTHALEIN PO4

FREE PHENOLPHTHALEIN

• OTHER METHODS:1. Bodansky

2. Jones

3. Reinhart

4. Bowers and Comb

• Pronounced elevations (5 or more time Pronounced elevations (5 or more time Normal)Normal) Bile duct obstruction (intrahepatic or extra)

Biliary cirrhosis

Paget’s disease

Osteogenic sarcoma

Hyperparathyroidism

• OTHER METHODS:1. Bodansky

2. Jones

3. Reinhart

4. Bowers and Comb

• Pronounced elevations (5 or more time Pronounced elevations (5 or more time Normal)Normal) Bile duct obstruction (intrahepatic or extra)

Biliary cirrhosis

Paget’s disease

Osteogenic sarcoma

Hyperparathyroidism

• Moderate elevation (3-5 times Normal)Moderate elevation (3-5 times Normal)

Granulomatous or infiltration dses of liver

IM

Metastatic tumor in bone

Metabolic bone disease (rickets, osteomalacia)

• Slight elevation (up to 3 times Normal)Slight elevation (up to 3 times Normal)

Viral hepatitis

Cirrhosis (intestinal isoenzyme often present

Healing fractures

Pregnancy (placental isoenzyme)

Normal growth patterns in children

ACP (EC 3.1.3.2, optimum pH6.0)

*investigation of RAPE CASES*investigation of RAPE CASES-Can persist for up to 4 days in vaginal -Can persist for up to 4 days in vaginal

washingswashings

*Citric acid acidification of serum = *Citric acid acidification of serum = stabilizes ACPstabilizes ACP

2 FORMS OF ACP:ACID PHOSPHATASE

RBC PHOSPHATASE

PROSTATIC, SPECIFIC FORM

NON-PROSTATIC,NONSPECIFIC

INHIBITED BY L-TARTRATE

INHIBITED BY FORMALDEHYDE AND CUPRIC IONS

METHODS OF ACP MEASUREMENT:

METHOD SUBSTRATE END PRODUCT

1. GUTMAN AND GUTMAN PHENYL PO4 INORGANIC PO4

2. SHINOWARA PNPP P-NITROPHENOL

3. BABSON, READ AND PHILIPS

ALPHHA-NAPHTHYL PO4 A-NAPHTHOL

4. ROY AND HILLMAN THYMOLPHTHALEIN MONOPO4

FREE THYMOLPHTHALEIN

THYMOLPHTHALEIN MONOPHOSPHATE

- most specific substrate

-substrate of choice for quanti. endpoint rxn.

ALPHA-NAPHTHYL PHOSPHATE

-preferred for continuous monitoring methods

THYMOLPHTHALEIN MONOPHOSPHATE

- most specific substrate

-substrate of choice for quanti. endpoint rxn.

ALPHA-NAPHTHYL PHOSPHATE

-preferred for continuous monitoring methods

AMINOTRANSFERASES• Catalyze the reversible transfer of an amino group reversible transfer of an amino group

between an amino acid and an alpha-keto acidbetween an amino acid and an alpha-keto acid• both ALT and AST require pyridoxal phosphate as a pyridoxal phosphate as a

cofactorcofactor• rich in the liver (organ for protein synthesis)• ALT has high specificity for liver damageALT has high specificity for liver damage • AST- large amount in liver, myocardium & skeletal AST- large amount in liver, myocardium & skeletal

muscle; moderate in RBCsmuscle; moderate in RBCs• hepatocytes contain 3-4x more AST than ALT

CHARACTERISTIC AST ALT Present in tissues other than liver

More in heart than in liver; also in the skeletal muscle, kidney, brain

Relatively low concs. In other tissues

Location in hepatocyte Mitochondria & cytoplasm

Cytoplasm only

Reference range in adult blood

10-40 IU/L 5-35 IU/L

Change with acute inflammatory damage

Moderately sensitive Extremely sensitive

Change with primary or secondary neoplasm

Substantial rise Moderate or no rise

Change with cirrhosis Moderate rise Mild or moderate rise

Change with myocardial infaret

Substantial rise Mild or moderate ruse

SGOT/AST (EC 2.6.1.1)

SGPT/ALT (EC 2.6.1.2)

ORGAN AFFECTED HEART LIVERSUBSTRATE ASPARTIC ALPHA-

KETOGLUTARIC ACIDALANINE ALPHA-KETOGLUTARIC ACID

END PRODUCTS GLUTAMIC ACID GLUTAMIC ACID + PYRUVIC ACID

COLOR DEVELOPER 2,4-DNPH 2,4-DNPH

COLOR INTENSIFIER 0.4 NaOH 0.4 NaOH

METHODS REITMAN AND FRANKEL

REITMAN AND FRANKEL

INCUBATION PPERIOD 1 HR. @ 37C 30 MINS. @ 37C

AMYLASE (Alpha-1,4-Glucan-4-Glucohydrolase EC 3.2.1.1)

• Also diastase (cleared in the urine)diastase (cleared in the urine)• found in salivary glands & pancreassalivary glands & pancreas• pancreatitis pancreatitis causes amylase to increase in blood• acute- serum AMS rise within 6-24 hrs, back to acute- serum AMS rise within 6-24 hrs, back to

normal in 2-7 daysnormal in 2-7 days

2 ISOENZYMES:2 ISOENZYMES:

1.1. Salivary Amylase (Ptyalin) Salivary Amylase (Ptyalin)

2.2. Pancreatic Amylase (Amylopsin)Pancreatic Amylase (Amylopsin)

pronounced elevation (5 or more pronounced elevation (5 or more times normal)times normal)

Acute pancreatitisPancreatic pseudocystMorphine administration

Moderate elevation (3-5 times Moderate elevation (3-5 times normal)normal)

Pancreatic CA affecting head of pancreas (late manifestation)MumpsSalivary gland inflammationPerforated peptic ulcerIonizing rediation

METHODS OF AMYLASE MEASUREMENT (based on the disappearance of starch)

METHODS PRINCIPLE

SACCHAROGENIC -Measures the amount of reducing sugars produced by the hydrolysis of starch by the usual glucose methods

AMYLOCLASTIC -Measures amylase activity by following the decreases in substrate concentration

CHRONOMETRIC -Measures the time required for amylase to hydrolyze completely all the starch present in a reaction mixture. Endpoint is reached when there is absence of any substrate capable of producing the BLUE starch iodine color

AMYLOMETRIC -Measures the amount of starch hydrolyzed in a fixed period of time using the intensity of the BLUE starch iodine color

LIPASE (Triacylglycerol Acylhydrolase EC 3.1.1.3)

-Hydrolyzes the ester linkages of fats to produce ALCOHOL AND FATY ACIDS-OLIVE OIL (50%) = SUBSTRATE-found almost exclusively in the pancreas -found almost exclusively in the pancreas (specific for pancreatitis)(specific for pancreatitis)

-not cleared in the urine & thus may -not cleared in the urine & thus may remain elevated even after parallel release remain elevated even after parallel release of amylase goes back to normalof amylase goes back to normal

METHODS OF LIPASE MEASUREMENT:

METHODS OF LIPASE MEASUREMENT:

Distribution of Diagnostically Important Enzymes

Distribution of Diagnostically Important Enzymes

Distribution of Diagnostically Important Enzymes