Enzymes and Cardiac Markers

Enzymes: Background & Objectives

• Definition of an enzyme and the different classes.

• Tissue sources of the different enzymes.

• Enzymes’ diagnostic significance and assays to measure them; limitations of the assays.

• Importance of their measurement.

• Use of enzymes in differential diagnosis of disorders: Cardiac, Hepatic, Bone, Muscle, Malignancies, acute pancreatitis

• Evaluation of patient’s serum enzyme levels in relation to disease states. – Observe levels

Enzymes

• Definition: a protein that catalyses one or more specific biochemical reactions.

• Enzyme activity measured in the body (body fluids) and changes monitored in either substrate or product concentrations.

• ↓ enzyme concentration = ↑ substrate utilisation.

• Better to measure enzyme activity than enzyme protein concentration directly = expensive!

• Enzyme activity is more prone to analytical variation unlike protein concentrations.

• Enzyme concentration in cell > plasma.

• Normal enzyme result = BALANCE:

  • Rate of synthesis

  • Released into plasma

  • Rate of clearance

• Kinetic/Spectrophotometry: specific absorbance depending on methodology

Enzyme Imbalances

• Enzyme activity may be:

  • INCREASED

    • Proliferation of cells

    • ↑ Cell turnover or damage

    • ↓ Clearance from plasma

  • DECREASED (Lower than normal):

    • ↓ Synthesis (occasional)

    • Congenital deficiency/ low biology

Enzyme Activity Measurement

Diagnostic precision of plasma enzyme analysis may be improved by:

• Estimating more than one enzyme

• Isoezyme determination

• Serial enzyme estimation

• Look at other analytes e.g. decreased rate of clearance due to renal disease

• Patient history e.g. alcohol (non-specific cause of raised levels)

• Analytical factors affecting result: concentration of substrate & product, PH, temperature of reaction, type of buffer, presence of activators/inhibitors.

• Physiological factors:

  • Age: GOT, ALP

  • Gender: GGT Men > Women

  • Physiological conditions:

    • ALP & Pregnancy

    • CK/transaminases & exercise/parturition

Major Enzymes of Clinical Significance

• ACP: Prostatic carcinoma

• ALT: Hepatic disorders

• ALP: Bone disorders, Hepatic disorders

• Amylase & Lipase: Acute pancreatitis

• AST: Myocardial Infarction, Hepatic disorders, skeletal muscle disorders

• CK: MI, Skeletal muscle disorders

• GGT: Hepatic disorders

• G-6-PD: Drug-induced haemolytic anaemia

• LDH: MI, Hepatic disorders, Carcinomas

• Lipase: Acute pancreatitis

• Cholinesterase: Organophosphate poisoning, Genetic variants

Enzymes: AST/GOT

• Tissue of origin:

  • Present in high concentrations in: Heart, skeletal, liver, kidney, erythrocytes.

• Reasons for ↑ GOT levels:

  • Artefactual: haemolysis in vitro; delayed separation

  • Physiological: neonatal period

  • MI (↑↑GOT – heart!)

  • Acute viral or toxic hepatitis (↑↑GOT)

  • Cirrhosis

  • Infectious mononucleosis

  • Cholestasis with jaundice

  • Skeletal muscle disease (after trauma or surgery)

  • Severe haemolytic episodes

  • Etc…

Enzymes: ALT/GPT

• Tissue of origin:

  • Present in: Liver > skeletal, kidney, & heart.

• Reasons for ↑ GPT levels:

  • Circulatory failure with ‘shock’ &hypoxia (↑↑GPT)

  • Acute viral or toxic hepatitis (↑↑GPT)

  • Cirrhosis

  • Infectious mononucleosis

  • Cholestasis with jaundice

  • Skeletal muscle disease

  • Etc…

Enzymes: LDH

• Tissue of origin:

  • Present in high concentrations in: Heart, skeletal, liver, kidney, brain, erythrocytes [NON-SPECIFIC].

• Reasons for ↑ LDH levels:

  • Artefactual: in vitro, delayed separation of plasma (unstable).

  • MI (↑↑LDH)

  • Some haematological conditions: e.g. megaloblastic anaemia, acute leukaemia, lymphomas (↑↑LDH); thalassaemia, myelofibrosis, haemolytic anaemias, pernicious anaemia.

  • Renal infarction & kidney transplants (↑↑LDH)

  • Viral hepatitis

  • Skeletal muscle disease

  • Infectious mononucleosis.

  • Etc…

Enzymes: LDH - Isoenzymes

• 5 Isoenzymes: Electrophoresis [Travel: LD1 > LD5].

• Since elevated in a number of diseases = non-specific; use isoenzymes and observe in conjunction with other analytes.

• Fraction present: LD-2 > LD-1 & LD-3 > LD4 > LD-5.

• LD-1: most significant in MI, measure in conjunction with HBDH (alpha-Hydroxybutyrate dehydrogenase) & CK
  Thus, LD-1 % becomes higher than LD-2.
  To analyse: ELECTROPHORESIS or Immunoinhibition or Differences of Substrate affinity

Enzymes: LDH - Isoenzymes

• Isoenzyme Composition and Location:

  • LDH1 (H4): Myocardium, RBC, kidney

  • LDH2 (H3M₁): Myocardium, RBC, serum, kidney

  • LDH3 (H2M2): Kidney, Skeletal muscle

  • LDH4 (H₁M3): Kidney, Skeletal muscle

  • LDH5 (M4): Skeletal muscle, Liver

Enzymes: CPK/CK

• Tissue of origin:

  • Present in : Heart, skeletal muscle, brain, & smooth muscle.

  • Associated with ATP generation in contractile muscle or transport systems; ↑ levels in males > women

• Reasons for ↑ CK levels:

  • Artefactual: in vitro haemolysis, delayed analysis

  • Physiological: neonatal period, after parturition, intensive exercise, bed-ridden

  • Circulatory failure with ‘shock’ & hypoxia (↑↑CK)

  • MI (↑↑CK)

  • Muscular dystrophy & rhabdomyolysis (↑↑CK)

  • Muscle injury & physical exertion (incl: muscle cramps, epileptic fits)

  • Intramuscular injection

  • Alcoholism

  • Etc

Enzymes: CPK/CK

• CK consists of 2 subunits: M & B → 3 isoenzymes: BB (CK-1), MB (CK-2) & MM (CK-3)

  • CK-MM: detectable in plasma of normal subjects.

  • CK-MB accounts for app. 35% of total CK activity in cardiac muscle & <5% in skeletal ∴ ↑ CK-MB after MI! [>6%= strong indications of AMI}

  • Isoenzymes usually measured = ‘routine’ Total CK

  • In cardiac cases = e.g. after MI measure CK-MB (↑).

  • CK-BB: associated with a number of conditions as well as during parturition; BUT measurement NOT of diagnostic benefit!

Enzymes: ⍺-amylase

• Catalyses breakdown of starch and glycogen to glucose; filtered in urine (sometimes diagnostic).

• Tissue of origin:

  • Present in : Pancreatic Juice (pancreas), saliva (salivary glands), gonads, fallopian tubes, skeletal muscle, adipose tissue.

• Reasons for ↑ ⍺-amylase levels:

  • Acute pancreatitis

  • Diabetic ketoacidosis

  • Pancreatic tumours

  • Acute abdominal disorders: acute cholecystitis, intestinal obstruction, abdominal trauma, ruptured ectopic pregnancy

  • Salivary gland disorders: Sjögren’s, mumps, salivary calculi

  • Etc…

Enzymes: ALP

• Tissue of origin:

  • Present in : Osteoblasts (BONE), hepatobiliary tract (LIVER) > intestinal walls, renal tubules, placenta

• Reasons for ↑ Total sALP levels:

  • Physiological: last trimester of pregnancy, children (growth spurt), gradual increase with age (liver ALP), following osteoporotic fractures.

  • Bone disease: rickets/osteomalacia, Paget’s, 1° malignant deposits in bone, osteogenic sarcoma, 1° & 2° hyperparathyroidism.

  • Liver disease: Cholestasis, tumours, granulomas (hepatic infiltration).

  • Malignancy – any with bone or liver involvement or direct tumour production.

• Reasons for ↓Total sALP levels:

  • Arrested bone growth; osteoporosis [lower levels in old age (↓bone mass, ↓OB)].

  • Hypophosphatasia: autosomal dominant associated with rickets/osteomalacia.

Enzymes: ALP Isoenzymes

• Isoenzymes originate from:

  • BONE

  • LIVER

  • INTESTINES

  • PLACENTA

• Separated by Electrophoresis or measured specifically

• Limitations:

  • Difficult to interpret as difference between Total and specific isoenzyme is minimal.

  • Expensive!

• Placental isoenzymes are heat-stable as opposed to the others.

Enzymes: GGT

• Tissue of origin:

  • Present in: Liver, kidneys, pancreas, prostate. [MEN > WOMEN]

• Reasons for ↑ GGT levels

  • Induced enzyme synthesis BUT NO cell damage → drugs & alcohol.

  • Cholestatic liver disease: GGT levels parallel ALP

  • Hepatocellular damage: e.g. viral hepatitis. (N.B. Transaminases more sensitive indicator; elevated before).

  • ↑↑↑GGT as opposed to transaminases:

    • Alcoholic hepatitis or chronic alcohol intake

    • Anticonvulsant drugs

    • Cholestatic liver disease

• Limitation: A slight or moderately raised GGT is difficult to interpret. → Obtain patient history!

Table 13.3 Abnormalities in liver function tests that may help to differentiate cause.

• Hepatocellular damage

  • Cholestasis

  • Cirrhosis

  • Tumours

Enzymes: ACP

• Tissue of origin:

  • Present in: Prostate > liver, erythrocytes, platelets, bone.

• Reasons for ↑ ACP levels:

  • Prostatic carcinoma (↑ no. of prostatic acid phosphatase-containing cells).

• Benefits of measurement: To MONITOR Prostate cancer

• Limitations:

  • Normal levels in Small tumours or too undifferentiated to synthesize enzyme. not good for diagnosis → Replaced by PSA

  • Rectal examination may increase levels; levels fall after a week.

  • Only serum used as heparin inhibits ACP.

  • Separated immediately as enzyme is unstable.

  • Haemolysis avoided → ACP released from RBCs.

• Why needed? → To measure the prostatic fraction! [ONLY IMPORTANCE!]

Myocardial Infarction

• Symptoms, physical examination, and patient history, But there are many causes of chest pain other than AMI

• EKG … only 50% reliable - may be normal even during AMI

• Laboratory tests (Troponin I, CKMB, Myoglobin, BNP)

Myocardial Infarction: IMP Notes

• Diagnosis: Look at Electrocardiograms & Biomarker levels

  • Before CK, LDH, HBDH, GOT were used.

  • Replaced with: Troponin I & CK-MB

• Turnaround Time:

  • Take into account TIME ELAPSED since the suspected infarct…. TESTED IMMEDIATELY!

  • Blood specimen: AT LEAST 4 HOURS AFTER ONSET of chest pain

  • NOTE: ↑CK-MM may be elevated due to recent intramuscular injection, exercise or surgery →GET PATIENT HISTORY

• Levels:

  • Degree of rise is a very rough indicator of the size of the infarct BUT of limited prognostic value (depends more on site rather than size)

  • A second rise after depression in values = extension of damage.

Myocardial Infarction: Tests

• Kinetics of common cardiac markers in blood following AMI

  • Marker, Initial Rise, Peak, Return to Baseline

  • Myoglobin: 2-3 h, 6-9 h, 18-24 h

  • CK-MB: 3-8 h, 10-24 h, 3-4 d

  • Troponin (large MI): 4-6 h, 10-24 h, Up to 7 d or more

Myocardial Infarction: Troponin

• Why TROPONIN??

  • troponins have nearly absolute myocardial tissue specificity… No elevations due to other conditions!

  • Contractile protein associated with cardiac and skeletal tissue

  • Reflect even microscopic zones of myocardial necrosis

  • Cardiac Troponin values also measured as an early & specific marker of acute MI.

• Two forms exist:

  • Troponin I

  • Troponin T

• Troponin I from cardiac tissue has a unique antigenic structure that differentiates it from skeletal Troponin and facilitates its measurement

• Of all the cardiac markers, Troponin I is the most specific for cardiac injury & necrosis

Myocardial Infarction: Myoglobin

• Soluble haem protein

• Present in all muscle cells, cardiac and skeletal

• Plasma myoglobin is elevated in various forms of muscle damage - surgery, strenuous exercise, degenerative muscle diseases and physical trauma

• Myoglobin can also be elevated from decreased renal clearance

• Consequently, the use of myoglobin is no longer necessary for acute MI. In addition, myoglobin is not specific for the diagnosis of myocardial necrosis

Summary - Heart Disease

• Troponin T or I are the most important biochemical markers of myocardial infarction.

• The myoglobin level may rise rapidly after myocardial infarction, but this finding is relatively nonspecific.

• Creatine kinase (CK-MB) is used less often.

• Alanine transaminase and lactic dehydrogenase are also used less often and are relatively nonspecific.

Myocardial Infarction: B-Type Natremic Protein (BNP)

• Small peptides produced by the heart continuously but their production increases when the heart muscle is stretched & pumps more blood

• Increased plasma BNP is associated with Congestive Heart Failure (CHF)… released in response to pressure in the heart

  • THUS, associated with CHRONIC heart conditions

• CHF is one of the most common reasons for hospitalization in patients >70 year of age

• BNP assays are often ordered with AMI markers to differentiate between AMIs and CHF

• BNP is normal in AMI

BNP or NT-proBNP

• Since BNP and NT-proBNP are elevated in patients with HF, both are useful adjuncts to clinical evaluation

• there was relatively low diagnostic concordance and correlation between BNP and NT-proBNP using the current cutoffs for HF.

• chronic kidney disease had a profound negative impact on concordance between the two tests

• measurable concentrations of NT-proBNP are higher in plasma than BNP necessitating different clinical cut-points. For example, the accepted rule out cut-points for acute HF for BNP is 100 pg/mL and for NT-proBNP is 300 pg/mL.

• There are multiple, well known issues affecting immunoassay measurement for BNP and NT-proBNP, including differences in:

  • protein glycosylation,

  • half-lives,

  • renal clearance,

  • biochemical diversity in HF patients, and

  • variable reactivity of antibodies with the precursor pro-BNP

Muscle Disease

• Muscle dystrophies = ↑CK & transaminases.

• Enzyme activity levels are highest:

  • Early stages of the disease – later stages = muscle wastage ↓enzymes

  • Following muscular activity with blood taken exactly after rest

  • Newborns

• Specimen collection:

  • Late in the day after ordinary physical activity

  • Not during pregnancy

  • Not after severe exercise or intramuscular injection (>48hrs).

Enzyme activity levels

• Heart disorders

• Muscle diseases

• Hepatic disorders

• Bone disorders

• Acute pancreatitis

• Malignancy e.g. prostate, bone, liver:

  • diagnostic,

  • prognostic,

  • indication of spread & secondary deposits,

  • monitoring of treatment.

• Haematological e.g. megaloblastic anaemia, leukaemias