FE A. BARTOLOME, MD, DPASMAP Dept. of Pathology & Laboratory Diagnosis Our Lady of Fatima University.

FE A. BARTOLOME, MD, DPASMAPFE A. BARTOLOME, MD, DPASMAP

Dept. of Pathology & Laboratory DiagnosisDept. of Pathology & Laboratory Diagnosis

Our Lady of Fatima UniversityOur Lady of Fatima University

• protein molecules

• catalyze chemical reactions without themselves being altered chemically

• contained primarily within cells

• essential enzymes present in virtually all organs but with slightly different forms in different locations isoenzymes

• classified according to biochemical functions

• unit of enzyme activity:

1 IU = transform 1 mol of substrate/minute

1 SI (katal) = transform 1 mol substrate/second

ENZYMES

Indicators of Cardiac Injury

Creatine kinase (CK)

• creatine phosphokinase (CPK)

• catalyze transfer of a PO4 group between creatine PO4 & ADP to form creatine + ATP

• requires magnesium as cofactor

• dimeric with pair of two different monomers M & B

• 3 isozymes: CK1 (BB), CK2 (MB), CK3 (MM)

• M and B subunits antigenically distinct proteins encoded by different genes

• primary tissue sources:

1. Brain, smooth muscle, prostate, thyroid, gut, lung CK-BB

2. Cardiac muscle – MB (20-30%) & MM (70-80%)

3. Skeletal muscle – MB (1-2%) & MM (98-99%)

4. Plasma – predom. MM with < 6% MB

• relatively small molecular size allows leakage out of ischemic muscle or brain cells


• reference ranges in serum affected by:

1. Amount of lean muscle mass

Thin, sedentary = 30 – 50 U/L

Muscular, exercising regularly = 500 – 1000 U/L

2. Age – in neonates, CK-MB 5-10% of total CK

3. Gender

4. Race – Africans 30% higher than Europeans

5. Muscle activity – direct relationship between intensity of exercise and CK level


• persons exercising periodically & at usual intensity levels with lower CK than those who do not exercise at all

• decrease with severe inactivity

• short-term strenuous exercise 10-100 fold increase

• marathon runners up to 2000 U/L as resting value


• released from damaged muscles: CK, AST, LD, myoglobin

Myoglobin >> CK >> AST and LD

• released during ischemia, injury or inflammation

• also increased in:

1. Chronic myopathies

2. Chronic renal failure

3. Acute respiratory exertion – respiratory muscles with more CK than other muscles

Diagnostic Applications

CM-MM

• Brain trauma or brain surgery

1. Injury to smooth muscles (e.g. intestinal ischemia)

2. Patients with malignancies, esp. prostate cancer, small cell lung CA, intestinal malignancies synthesize B subunit

3. Transient increase after cardiac arrest reflect cerebral ischemia

CK-BB


• primary clinical use: detection of acute MI

Following MI:

Total CK – 98% sensitive but 68-85% specific; peak value 18-30 hrs; duration 2-5 days; level 5-10x normal

CK-MB – rise proportional to extent of infarction; appears in serum within 6 hrs after AMI; peak value 12-24 hrs; duration 1.5-3 days persistence indicates extension or infarction or re-infarction

CK-MB


Normal: 24 – 170 U/L (women) 24 – 195 U/L (men)

• Marked elevation (> 5x normal)

1. After trauma from electrocution, crush injury, convulsion, tetany, surgical incision or IM injection

2. Athletic individuals – inc. muscle mass & inc. release during strenuous activity

3. Muscular dystrophies

4. Chronic inflammation of muscle (dermatomyositis or polymyositis)

Total Serum CK


• Mild or moderate elevation (2 – 4x normal)

1. Hyper- or hypothermia

2. Hypothyroidism

3. After normal vaginal delivery – BB isoenzyme from myometrial contractions

4. Reye’s syndrome


Total Serum CK

Forms that migrate electrophoretically in positions different from standard ones

1. Adenylate kinase

Catalyze formation of ATP & AMP from ADP

Released from erythrocytes

2. Macro CK type 1 – complex of CK (BB) with antibody (IgG) mistaken for CK-MB; no known clinical significance

3. Macro CK type 2 – oligomeric variant of CK; mitochondrial

If (+) in serum – poor prognostic sign

(+) in patients with malignancies & moribund patients

Atypical Isoenzymes

• Zinc-containing; part of glycolytic pathway

• Catalyze conversion of lactate to pyruvate using NAD+ as cofactor

CH3 CH3

HCOH + NAD+ C = O + NADH + H+

COOH COOH

• Tissue source: present virtually in all tissues cytoplasm of all cells and tissues in the body

• Tetramers with 4 subunits of 2 possible forms: H (heart) and M (muscle)

Lactate dehydrogenase (LD)

• Five isoenzymes:

LD1 & LD2 – high in heart muscle, erythrocytes, kidney

LD4 & LD5 – high in skeletal muscle & in liver

• Normal pattern in serum:

LD2 > LD1 > LD3 > LD4 > LD5

• Highest in newborns and infants; values do not change with age in adults

• No gender difference

Lactate dehydrogenase (LD)

• Total LD activity: increase in any disease state where there is cell damage or destruction non-specific correlate with AST, ALT and CK

• Markedly inc. LD with normal or minimally inc. AST, ALT & CK damage to biochemically simple cells (e.g. rbc, wbc), kidney, lung, LN or tumors

• Inc. LD & CK; inc. AST > ALT cardiac or skeletal muscle injury

• AST & ALT inc. > LD transiently in liver disease (toxic or ischemic liver injury)

DIAGNOSTIC APPLICATIONS:

• Myocardium normally with LD1 > LD2 similar to rbc

• Acute myocardial infarction:

Inversion or flipped LD1/LD2 ratio to a value > 1.0 in serum stay flipped for several days

Levels inc. after 12-24 hrs, peak (2-10x normal) at 48-72 hrs, return to normal after 8-10 days

• used to confirm diagnosis of MI when CK isoenzyme analysis equivocal or after total CK & CK-MB release has returned to normal

Myocardial damage –

• Total LD used to estimate tumor mass including metastases

LD1 or LD2 inc. in germ cell tumors (seminoma & dysgerminoma) – serve as tumor marker

• Flip LD1/LD2 ratio:

Extreme exercise

Acute myocardial infarction

Hemolytic anemia

Megaloblastic anemia

Renal cortical disease (renal infarct, renal cell CA)

Other Applications

• Inc. LD2, 3 and 4 – malignancy & large tumor burden

• Inc. LD3 & 4, dec. LD1 & 2 – WBC tumors (leukemia, lymphoma, MM), pulmonary disease

• Inc. LD4 & 5 – skeletal muscle injury, ischemic or toxic hepatic injury

• Isomorphic pattern – inc. total LD, normal isoenzymes with “tombstone” pattern (relative amounts of isoenzymes the same) diffuse tissue damage accompanied by shock or hypoxemia

Other Applications

Myoglobin

• Small; functions in storage and transfer of O2 from Hgb in the circulation to intracellular respiratory enzymes of contractile cells

• With greater affinity for oxygen than Hgb

• Only one molecular form

• One of the first to diffuse out of ischemic muscle cells, even before CK

• Cleared from circulation by kidneys

• Measurement in serum with high sensitivity for muscle injury, including acute MI measure by immunoassay

Myoglobin

• In normal individuals,

Levels related to muscle mass and activity

Males > females

Africans > Europeans

Increase with increasing age due to decreasing GFR

Troponins

• Bind tropomyosin and govern excitation-contraction coupling

• Three subunits

1. Troponin C (TnC) – calcium-binding subunit

2. Troponin I (TnI) – bind to actin inhibitory

3. Troponin T (TnT) – bind to tropomyosin

• TnI and TnT with unique forms expressed in myocardial cells but not in other muscle types presence of cTnI or cTnT in serum highly specific for myocardial injury

• cTnT

84% sensitivity for MI 8 hrs after onset of symptoms

81% specificity for MI; 22% specificity for unstable angina

• cTnI

90% sensitivity for MI 8 hrs after onset of symptoms

95% specificity for MI; 36% specificity for unstable angina

Troponins

• Cardiac troponins released in two phases:

1. Initial damage (acute MI) – leave myocardial cells enter circulation the same time that CK-MB does peak at 4-8 hrs

2. Sustained release from intracellular contractile apparatus – occurs up to days after acute event

• First appear in circulation ff. myocardial injury slightly later than when myoglobin enters the blood rises after 3-6 hrs peaks at ~ 20 hrs

Troponins

• General advantages:

1. cTnT and cTnI are released only following cardiac damage.

2. Unlike CK & CK-MB, cTnT and cTnI are present , and remain elevated, for a long time cTnI detectable up to 5 days & cTnT for 7-10 days following MI

3. cTnT and cTnI are very sensitive.

Troponins

• General disadvantages:

1. Elevation can occur as a result of causes other than MI myocarditis, severe cardiac failure, cardiac trauma, pulmonary embolus with cardiac damage

2. Failure to show a rise in cTnT or cTnI does not exclude the diagnosis of ischemic heart disease.

3. Both may be elevated in patients with chronic renal failure with sustained levels of elevation.

Troponins

• Measured in serum by immunoassay

• Ideal time to check is between 6 and 9 hours from onset of symptoms

If onset of symptoms indistinct – take sample on admission, 6 – 9 hrs after and at 12 – 24 hrs after admission

Troponins

Other enzymes useful in clinical diagnosis

Acid phosphatase (ACP)

• Optimal activity: pH 5.0

• Tissue source:

Common to many tissues, esp. prostate

Small amounts in rbc, platelets (during clot formation), liver and spleen

Human milk and seminal fluid (very concentrated)

• Prostatic ACP distinguished from others using thymolphthalein monophosphate highly specific for prostatic ACP

• Major applications:

1. Evaluation of prostatic CA (metastatic & local growth)

Not elevated in CA confined within prostate, BPH, prostatitis or ischemia of prostate

2. Medicolegal evaluation of rape – vagina with little or no ACP

• Measured by radioimmunoassay acidify serum with citric acid to stabilize ACP activity

Acid phosphatase (ACP)

• Widely distributed along surface membranes of metabolically active cells

• Encoded for by four different genes expressed in:

1. Placenta

2. Intestines

3. Germ cell and lung

4. Tissues including bone, liver, kidney & granulocyte

• Very high activity in bone, liver, intestine, kidney, wbc and placenta

Alkaline phosphatase (ALP)

• Methods for distinguishing ALP isoenzymes:

1. Heat fractionation – easiest & most common; heat serum sample at 56oC x 15 min. then compare with unheated sample

Bone ALP extremely labile retain 10-20% of original activity

Liver & placental ALP heat stable liver ALP 30-50% retained, placental ALP with all retained

2. Chemical inhibition

Urea – block placental ALP

Phenylalanine – block liver & bone ALP

3. Electrophoresis - definitive

Alkaline phosphatase (ALP)

DIAGNOSTIC APPLICATIONS

Derived from epithelial cells of biliary tract excreted by bile into intestine

• Used for establishing diagnosis in jaundice

• Pronounced increase (> 5x)

Intra- or extrahepatic bile duct obstruction

Biliary cirrhosis

• Moderate increase (3-5x normal) : granulomatous or infiltrative liver disease

• Slight increase (up to 3x normal) : viral hepatitis, cirrhosis

Liver ALP

Elevation part of osteoblastic growth

• Pronounced increase:

Paget’s disease

Osteogenic sarcoma

Hyperparathyroidism

• Moderate increase: metastatic tumors in bone; metastatic bone disease (rickets, osteomalacia)

• Slight increase: healing fractures; normal growth patterns in children

Bone ALP

Placental ALP

• With oncofetal form turned on and expressed by tumor cells in adults called Regan isoenzyme

• Slight increase in pregnancy

Intestinal ALP

• Inc. in inflammatory bowel disease (ulcerative colitis & regional enteritis)

• Secreted into the circulation after a meal in persons with blood type “O” and “B” inc. total ALP in non-fasting specimens

Renal ALP

• Normally excreted into urine from renal tubular cells

Granulocyte ALP

• Used as marker of granulocyte maturity in leukocytosis

• Lymphocytes infected with HIV release specific ALP fraction (band-10) surrogate marker for HIV infection in children

• Glycolytic enzyme split fructose-1,6-diphosphate into two triose phosphate molecules in glucose metabolism

• Distributed in all tissues

• Elevated in serum following:

1. Skeletal muscle disease or injury – reflect severity of dermatomyositis

2. Metastatic CA to liver 5. Hemolytic anemia

3. Granulocytic leukemia 6. Tissue infarction

4. Megaloblastic anemia

Aldolase

• Catalyze reversible transfer of an amino group between an amino acid and an alpha-keto acid

R R’ R R’

HCNH2 + C = O C = O + HCNH2

COOH COOH COOH COOH

• Requires pyridoxal phosphate (vitamin B6) as cofactor

Aminotransferase (Transaminase)

• “glutamate-pyruvate transaminase” (GPT)

• Rich amounts in hepatocytes with high specificity for liver damage

• Moderate amount: kidney, heart, skeletal muscle

• Small amount: pancreas, spleen, lung, red blood cells

Alanine aminotransferase (ALT)

• Inc. AST & ALT – excellent indicators of liver damage

• ALT increased in serum ff. acute MI 6 hrs after onset, peaks at 24-48 hrs, returns to normal in 3-4 days

• AST inc. in conditions that can be confused with acute MI or that may complicate or co-exist with acute MI:

1. Shock or circulatory collapse from any cause

2. Acute pancreatitis

3. Cardiac arrhythmias or ischemic insult that do not progress to infarction


• “gamma glutamyltranspeptidase

• Catalyze transfer of glutamyl groups between peptides or amino acids through linkage at a -COOH group important in transfer or movement of amino acids across membranes

• Large amounts in:

Pancreas and renal tubular epithelium

Hepatobiliary cells

Gamma glutamyltransferase (GGT)

• increased activity:

1. In urine – renal tubular damage

2. Hepatocellular & hepatobiliary diseases correlates better with obstruction & cholestasis than with pure hepatocellular damage “obstructive” enzyme


• GGT & alcohol

Alcohol induces microsomal activity inc. GGT synthesis indicator of alcohol use

GGT levels return to normal after 3-6 wks of abstention from alcohol test for compliance in alcohol-reduction programs


• GGT & drugs

Barbiturates, phenytoin & other drugs (acetaminophen) inc. microsomal activity of GGT

Potentially useful in drug treatment protocols


• digestive enzyme

• Acts extracellularly to cleave starch into smaller groups & finally to monosaccharides

• Major sources: salivary glands

exocrine pancreas

Amylase (Diastase)

• secretion stimulated by pancreozymin (cholecystokinin)

• enter duodenum at ampulla of Vater via sphincter of Oddi

• Low levels found in:

1. Fallopian tubes 3. Small intestine

2. Adipose tissue 4. Skeletal muscle

• readily cleared in urine

Pancreatic amylase

Acute Pancreatitis

• Levels rise within 6-24 hours remain high for a few days return to normal in 2-7 days

• Serum amylase normal but with suspicion of pancreatitis measure 24-hour urine amylase or serum lipase


Morphine administration

• Constrict pancreatic duct sphincter dec. intestinal excretion & inc. absorption in the circulation

Renal failure

• Failure to clear normally released amylase from the circulation no diagnostic significance


Malabsorption & liver disease

• (+) circulating complexes of amylase with a high MW compound such as Ig’s macroamylasemia prevent renal clearance

• no diagnostic significance


Tumors

1. serous ovarian tumors

• epithelium similar to FT produce cyst fluid with amylase appear in serum & urine

2. Lung CA

• ectopic production of amylase


Conditions Affecting

Serum Amylase

Pronounced Elevation (Pronounced Elevation (>> 5x normal) 5x normal)Acute pancreatitisAcute pancreatitisPancreatic pseudocystPancreatic pseudocystMorphine administrationMorphine administration

Moderate Elevation (3-5x normal)Moderate Elevation (3-5x normal)

Pancreatic CA (head of pancreas)Pancreatic CA (head of pancreas)

MumpsMumps

Salivary gland inflammationSalivary gland inflammation

Perforated peptic ulcerPerforated peptic ulcer

Ionizing radiationIonizing radiation

Angiotensin Converting

Enzyme (ACE)

• Action: convert angiotensin I to angiotensin II

• Site: lungs

• Main tissue source: macrophages & epithelioid cells

• Diagnostic use:

1. Active sarcoidosis – primary use

2. Other granulomatous diseases (e.g. TB)

3. Disorders of macrophage function (e.g. Gaucher’s disease & leprosy)

4. Normal adults & persons < 20 y/o – high levels

Cholinesterase

Pseudocholinesterase (CHS)

• Serum

• Synthesized in hepatocytes

• Able to act on a wider variety of choline esters

• Active at both high and low substrate concentration

• Inhibited by organophosphorous compounds (e.g. insecticides)

Cholinesterase

True Acetylcholinesterase (AcCHS)

• Erythrocytes and nerve endings

• Breaks down Ach into acetate and choline at post-synaptic sites

• Active at low substrate concentration & inhibited by high Ach concentration

• Inhibited by organophosphorous compounds

DIAGNOSTIC

APPLICATION

Serum CHS

• Decreased in organophosphate poisoning falls early after exposure & rises soon after exposure ceases used to document acute toxicity

• Decreased in hepatocellular disease

• Completely inactivates succinylcholine (muscle relaxant) in the circulation over a short interval (several seconds) (+) reversal of paralysis

• Normal activity inhibited by dibucaine

Erythrocyte AcCHS

• Inhibited less rapidly by organophosphate insecticides remains depressed even after serum CHS returns to normal used to document prior exposure

• Does not act efficiently on succinylcholine

DIAGNOSTIC

APPLICATION

Lipase

Alimentary lipase

• Cleave dietary TG’s into free fatty acid & glycerol

• Secreted by exocrine pancreas into the duodenum

• Found almost exclusively in pancreas highly specific

Lipase

Alimentary lipase

• Not cleared into the urine remain elevated after amylase has returned to normal

• Highest levels in acute pancreatitis

• Moderate increase: pancreatic CA

• Inc. after administration of morphine or cholinergic drugs (+) constriction of sphincter of Oddi

Lipase

Blood lipase

• Cleaves fatty acids from lipoproteins and clears chylomicrons from the circulation

• Bound to vascular endothelium membrane

• Released into plasma by administration of heparin occurs within minutes of IV heparin dose post-heparin lipolytic activity (PHLA)

Lysozyme (Muramidase)

• Low MW hydrolytic enzyme

• Catalyze breakdown of bacterial cell walls

• Sources: tears, saliva, sputum, granulocytes, monocytes

• Readily cleared into urine

• Quantitated by ability to lyse the bacterium Micrococcus lysodeikticus convert turbid suspension into a clear one

DIAGNOSTIC

APPLICATIONS

• Makedly elevated in serum & urine: acute monocytic & acute myelomonocytic leukemias

• Low levels: lymphoctic leukemia & chronic granulocytic anemia

• Serial levels used in detecting relapse in acute leukemias

ENZYME-ORGAN

ASSOCIATIONS

ORGANORGAN ENZYMEENZYME

LiverLiver Aminotransferases (AST, ALT)Aminotransferases (AST, ALT)Lactate DH (LD5)Lactate DH (LD5)Gamma-glutamyltransferaseGamma-glutamyltransferaseAlkaline phosphataseAlkaline phosphatase

HeartHeart Creatine kinase (MB)Creatine kinase (MB)Lactate DH (LD1 > LD2)Lactate DH (LD1 > LD2)Troponins I and TTroponins I and T

Skeletal muscleSkeletal muscle Creatine kinase (MM)Creatine kinase (MM)Lactate DH (LD5)Lactate DH (LD5)Aldolase Aldolase

BrainBrain Creatine kinase (BB)Creatine kinase (BB)

BoneBone Alkaline phophatase (heat labile)Alkaline phophatase (heat labile)

ProstateProstate Acid phosphataseAcid phosphatase

PancreasPancreas AmylaseAmylase

Lipase Lipase

ENZYME-ORGAN

ASSOCIATIONS

FE A. BARTOLOME, MD, DPASMAP Dept. of Pathology & Laboratory Diagnosis Our Lady of Fatima University.

Documents

total ck

lower ck

normal ckmb

lung ckbb

cardiac muscle mb

myoglobin myoglobin

skeletal muscle mb

creatine po