Important Enzymes and

Lipoproteins in Cardiac Diseases

Presented by: Mr Sony Peter
Madawalabu University
College of Medicine and Health Sciences
Department of Medicine
Biochemistry Unit
 Serum enzymes in heart diseases
• The investigation and interpretation of changes in serum enzymes
in disease is one of the most rapidly expanding fields in Medicine,
in clinical biochemistry discipline.

• Important assays that are carried out in myocardial infarction are:

1. Creatine Phosphokinase/ Creatine Kinase (CK)

2. Aspartate Transaminase
3. Lactate Dehydrogenase
4. Gamma- Glutamyl Transpeptidase
5. Pseudo-cholinesterase
6. Troponins
1. Creatine Kinase (CK)

This enzyme catalyses the following reaction:

Creatine- phosphate + ADP -----→ Creatine + ATP

 Found in high concentration in skeletal muscle, myocardium and

brain.
 After MI, serum value is found to:
 Increase after about 6 hours
 Reaches a peak level in 24- 30 hours
 Returns to normal level in 2-4 days

 Studies suggests that the serum CK activity is a more sensitive

indicator in early stage of MI
 CK is composed of 2 polypeptide chains, B and M, making up
of three forms: CK-MM, CK-MB and CK-BB
 Distribution of the three isomer forms of CK varies throughout
the tissues: CK-MM (Skeletal Muscle Tissue)
CK-MB (Cardiac Muscle Tissue)
CK-BB (Brain and Nerve Tissue)
• Historically MI was detected by looking CK isoenzyme CK-MB.
This marker is released into circulation from necrotic heart muscle.

As the heart muscle become damaged, this CK isoenzyme is released

into the blood stream and may be detected for 6-18 hours after onset
of AMI.

The window of detection is quite short, lasting no more than 12-18

hours after the heart attack occurred, because of protein
degradation mechanisms that eliminate the CK-MB from the blood.

Due to this short time frame, often the peak level of CK-MB is
missed, leaving in doubt whether a heart attack has occurred or this
is an indication of mild heart tissue damage or angina.
Reference Range

• Creatine Kinase:
Male: 46-171U/L

Female: 34-145 U/L

• CK-MB:
< 3.9% or < 5.0 µg/L
 2. Aspartate Transaminase (S-Glutamate Oxaloacetate
Transaminase)

• This is an intracellular enzyme involved in amino acid

metabolism. An increased level of this enzyme in blood
indicates necrosis or disease in the tissues.
• They catalyze transamination in mitochondria and cytoplasm
of heart.
• They can both, catabolize an amino acid to the
corresponding Keto acid and synthesize the former from
latter.
aspartate + alpha-ketoglutarate ↔ oxaloacetate + Glutamate
 Concentration of the enzyme is very high in myocardium.

 In AMI, serum activity rises sharply within the first 12 hours,

With a peak level at 24 hours or over and return to normal
within 3-5 days.

• Levels >350 IU/L should be considered fatal

• Elevations has been noted in absence of any ECG change
• Highest level of abnormal levels occurs on second day of
infarction
• Rise depends on the size of infarction
Reference Range

• Serum activity of s-GOT varies from 4-17 IU/L

3. Lactate Dehydrogenase

 LDH catalyzes the reversible conversion of pyruvic acid and

lactic acid.

 In AMI, serum activity rises within 12-24 hours,

 Attain peak at 48 hours, reaching about 1000 IU/L
 Returns gradually to normal from 8th to 14th day

 The peak rises in LDH is roughly proportionate to the extend

of injury to the myocardial tissue
 LDH elevation may persists for more than a week after CK
and S-GOT levels have returned to normal levels
• In particular, these enzymes and isoenzymes carry out their
metabolic functions within specific cells, and are released
into body fluids when those cells become diseased.

• Thus an increase in LD isoenzyme activity can indicate

leakage from cell due to cellular injury.

• The level of isoenzyme LD1 compared with LD2 has been

used to detect an AMI because of the high concentration of
LD in cardiac muscle fibres.

• LD isoenzymes begin to leak out of dying heart muscle cells

and are detectable in the serum by 12 to 24 hours following
a heart attack
• The appearance of more LD1 than LD2, also called ‘flipped
pattern’, is typical of cardiac muscle damage but is non-
specific since it also is associated with RBC cell haemolysis or
megaloblastic anaemia.

• The ‘flipped pattern’ pattern, in which LD1 > LD2, lasts up to

3-4 days after the heart attack
Reference Range

• Reference range of LDH in serum: 180-360 IU/L

• LD1: 14-26% of total LDH

4. ϒ-Glutamyl Transpeptidase

• G-GTP catalyzes the transfer of the ϒ-glutamyl group from

one peptide to another peptide or to an amino acid.

• Several investigators recently have demonstrated increase in

serum ϒ-glutamyl transpeptidase in acute myocardial
infarction. It will also get accumulated in atherosclerotic
plaques

• Increase serum activity is found to be late, peak activity

between 7th and 11th day and lasts as long as a month. Hence
it has been proposed as a useful test for MI in later stages.
Reference Range

• Men: 10-47 IU/L

• Women: 7-30 IU/l
5. Histaminase

• Normal plasma contains either very small amount of

histaminase or none at all, but considerable amount of
histaminase has been found in human heart muscle.

• Serum enzyme activity rises within 6 hours of MI and

persists for whole of first week.

• It helps in early diagnosis of MI even when ECG failed to

reveal.
• It also has a prognostic value as higher serum histaminase
levels were found to be associated with worse prognosis.
Reference Range

• The reference range of Histaminase activity in adult human: 0.12-0.76

PU/ml
6. Cholinesterase

• Cholinesterases are enzymes which hydrolyze esters of

choline to give choline and acid

They are of two types:

1. True cholinesterase: It is responsible for destruction of
acetyl choline at the neuromuscular junction and is found in
nerve tissues and RB cells.
2. Pseudo cholinesterase: It is found in various tissues such as
heart muscle and liver.
Raised activity is found within 12 hours or even as early as 3
hours found in some cases. Serum enzyme activity has been
considered as a sensitive index for determination of cellular
necrosis in myocardium.
7. Troponins

• Troponins are contractile proteins found within muscle fibres

that help regulate contractions.
• There are 3 troponins that work as a complex. They are:
Troponin C (ca- binding component)
Troponin I (Inhibitory component)
Troponin T (Tropomyosin- binding component)

 During the process of muscle necrosis, Troponins I and T are

released from the dying muscle fibres into the blood stream.
Increases in the concentration of troponins I and T above the
reference levels in serum indicate heart muscle fibre and
necrosis.
 Measurement of troponin assays has been a tremendous
boon to clinical diagnosis.
Troponins released from heart muscle remain in the blood
stream from 1 to 14 days after the onset of AMI.

 Troponins as cardiac markers, appear to have many

advantages primarily due to their quick release following
heart muscle damage and their longevity in the blood
stream following heart attack

 The risk of death from an Acute Coronary Syndrome (ACS) is

directly related to troponin level.
Reference Range
• Troponin I: 0.0- 0.05 ng/mL
• Troponin T: <0.01 µg/L
Lipoproteins and their relation to cardiac diseases

Serum Lipids and Lipoproteins

In human, principal lipids that have metabolic significance are as
follows:
1. TG
2. Phospholipids
3. Steroids
4. Fatty Acids
5. Glycerol

What are lipoproteins?

Water insoluble TG, saturated fatty acids, cholesterol and its esters
are transported in the plasma as water-miscible LIPOPROTEINS
(Conjugated Proteins)
• The four types of lipoproteins are:
1. Chylomicrons
2. VLDL
3. LDL
4. HDL

The Cholesterol form most associated with cardiovascular problems when in

excess LDL.

While LDL is considered harmful when in excess, the elevation of HDL is

viewed as a positive cardiovascular biomarker for a patient.

Elevated HDL has a beneficial effect for the vascular system due to the role
that HDL plays in the body.

HDL removes excess cholesterol from tissues and routes into the liver for
reprocessing or removal
Major functions of Lipoproteins
1. Chylomicrons:- transport mainly TG from either food or synthesised in
enterocytes, also small amount of PL, cholesterylesters and fat-soluble
vitamins from the intestine to liver, adipose tissues and muscles.
2. VLDL:- transport mainly endogenous TG, synthesized in hepatocytes,
from liver to extrahepatic tissues, mainly adipose tissues for storage.
3. LDL:- rich in cholesteryl esters, mainly transports cholesterol and its
esters from hepatocytes to extrahepatic tissues.
4. HDL:- transports cholesterol and its esters from extrahepatic tissues to
the liver.
Lipoprotein(a), is a unique among the lipoproteins. It is thought to be an
unusual type of LDL particle with an Apoprotein ‘a’ is covalently attached to
the Apoprotein B by a disulfide bridge. It is a modified form of LDL.
Because there is variability in the structure of apoprotein ‘a’, it is very
difficult to assay and detect all the possible variants.

Uniqueness of the Lipo(a), is also related to its similarity to plasminogen.

Plasminogen, when converted to plasmin in the blood, is instrumental in

degrading the incidental clots that are formed periodically in blood vessels.

When plasmin is not available, little clots become big ones and can lead to
blood vessel occlusions.

High lipo(a) level inhibit plasmin formation by attracting the plasminogen

activators and blocking their action
Thus, high levels of Lipo(a) are an independent risk factor for
patients. If patients have elevated levels of Lipo(a), their blood
tends to clot.

The apoprotein (a) chain contain 5 cystein rich domains known

as ‘Kringles’. The 4th kringle is homologous with fibrin-binding
domain of plasminogen, a plasma protein that dissolves blood
clots when activated.

Because of this structural similarity to plasminogen, lipo(a)

interferes with the fibrinolysis by competing with plasminogen
activation, plasmin generation and fibrinolysis.

This promotes the deposition of cholesterol as atherosclerotic

plaques.
 Primary and secondary hyperlipoproteinemia

Elevation of LDLs can result from inborn errors of metabolism, such as

enzyme or apoprotein deficiencies or from secondary causes or underlying
diseases.

Some of the diseases and medications that can cause hyperlipoproteinemia

includes DM, BP medications, nephrotic syndrome, chronic renal failure,
hepatic disorders including biliary obstruction.

Plaque build-up and blockage of blood vessels from excessive LDL level can
occur and lead to coronary heart diseases.
Hypoalphalipoproteinemia

The absence or a non-detectable level of apoprotein A-I is exhibited by a

severely decreased or absent level of HDL (Tangier’s disease/
hypoalphalipoproteinemia).

This condition leads to elevated LDL in the circulation with no viable HDL
action to remove cholesterol from the tissue.

Apoprotein A-I is the major apoprotein associated with HDL and is

necessary for the enzyme lecithin-cholesterol acyltransferace (LCAT) to
function. LCT joins a fatty acid to cholesterol, an alcohol, to make a
cholesterol ester. These cholesterol esters can be packed into the HDL
particles for transport to the liver, causing the HDL particle to go from disk
shape to spherical shape in the circulation.

When Apoprotein A-I is absent, LCAT is unable to function and esterification

of cholesterol is diminished.
Plaque build-up and blockage of blood vessels from excessive LDL levels can
occur and lead to coronary heart diseases. Heart ailments related to
hypoalphalipoproteinemia include atherosclerosis, MI and strokes.
Estimation of Cardiac Enzymes
 Electrolyte balance in the event of heart failure
Electrolytes or ions are charged particles in body fluids that help transmit
electrical impulses for proper nerve, heart and muscle function.

The number of positive ions called cations and negative ions called anions is
supposed to be equal. Anything that upsets this balance can have life
threatening consequences.

1. Na.
Na, the most abundant cation in the extracellular fluids, play a key role in
transmitting nerve impulses. It also helps maintaining serum concentration
or osmolality.

Hypernatremia, occurs when either too much water is lost or too much salt
is taken in. The elderly are particularly at risk for hypernatremia following
surgery or a fever because of volume depletion and because of diminished
thirst mechanism.
All patients on, hypertonic IV solutions or tube feeding are at risk as well.

Regardless of the causes, patients with hypernatremia may appear

tachycardic and lethargic. As their cells become more dehydrated, patients
may develop disorientation, weakness, irritability and muscle twitching.

Seizures, coma or death may result if hypernatremia is left untreated.

It can be treated by IV administration of isotonic solutions such as normal

saline or putting the patient on dialysis.

Hyponatremia, usually occurs when the body loses more Na than water or
when excess water dilutes the normal Na concentration.
Dilutional hyponatremia can be caused by excess fluid intake and conditions
such as congestive heart failures or syndrome of inappropriate ADH
secretion.

The neurological symptoms of hyponatremia are similar to those of

hypernatremia- lethargy and confusion with the possible addition of nausea
and vomiting resulting from cerebral edema.

It can be treated by ordering an increased intake of dietary Na.

Sever hyponatremia, however, is a medical emergency. Permanent

neurological damage can occur when serum Na falls below 110 mEq/L

Giving hypertonic saline (3%Nacl) solution is the treatment of choice, but

must be done with caution: A rapid serum sodium can cause fluid overload
and cardiac failure, as well as cerebral osmotic demyelination syndrome,
leading to paralysis and death.
2. Potassium
This electrolyte is the major intracellular cation. The 2% that is found in
extracellular fluid is crucial to neuromuscular and cardiac function.

Severe Hyperkalemia will slow cardiac impulse conduction, producing

changes in ECG.
Left untreated, the excess potassium will continue to supress conduction
until cardiac arrest occurs.

Severe hyperkalemia requires rapid action: stop any potassium

administration and give IV CaCl or calcium gluconate to stimulate
conduction.

Also order IV sodium bicarbonate, insulin and 50% dextrose or albuterol to

try to shift potassium out of the blood stream and back in to the cells.
Symptoms of hypokalemia include muscle weakness or tenderness, leg
cramps, drowsiness, confusion, loss of appetite and abnormal cardiac
conduction.

The lack of K makes cardiac muscle irritable, increasing the risk of

premature atrial or ventricular contractions that can trigger ventricular
tachycardia, which can progress to fibrillation and death.

Initial treatment of hypokalemia, especially when it is accompanied by

cardiac symptoms, focuses on replacing K.

This require great care: Given too rapidly, IV potassium can cause cardiac
arrest, so never administer it by IV push.
3. Magnesium
This cation is found primarily in the cells and is responsible for reactions
that involve muscle function, energy production and carbohydrate and
protein metabolism.

Renal failure is the major cause of hypermagnesemia. Other causes include

excessive intake of magnesium- containing antacids and laxatives and
conditions that produce acidosis such as diabetic ketoacidosis.

Signs of hypermagnesemia include lethargy, altered mental status that can

progress to coma, respiratory depression and muscle weakness.
Impaired cardiac conduction and contractibility produce bradycardia and
hypotension and can progress to a full cardiac arrest.

If patient’s renal function is normal, treatment may include diuretics to

promote magnesium loss.
Calcium gluconate given intravenously can help counteract muscle
weakness and improve cardiac function.
Patients with severe hypermagnesemia and renal failure my need
haemodialysis.

Hypomagnesaemia is common in critical ill patients and is associated with

high mortality rates.
Diuretic therapy, chronic alcoholism, cirrhosis, pancreatitis, can all cause
excessive Mg loss.

Hypomagnesaemia increases cardiac muscle irritability and the potential for

ventricular dysrhythmias, especially in patients with a recent MI.

Treatment focuses on giving the patient Mg either IV or oral form-as

ordered to return levels to normal. This is especially important in patients
recovering from an acute MI.
4. Calcium and Phosphorus
These electrolytes are inversely related in the blood. When calcium levels
are high, phosphorus levels are low and vice-versa.

Calcium is a cation with multiple functions, including transmitting nerve

impulses, maintaining cell wall permeability and activating the body’s
clotting mechanism.

It is also involved in contracting cardiac and smooth muscle, generating

cardiac impulses, mediating cardiac pacemaker function and forming bones
and teeth.

Phosphorus, the major intracellular anion, is necessary for energy

production in the cells and for carbohydrate, protein and fat metabolism, as
well.
They also helps maintain acid-base balance by buffering hydrogen ions.
Both hypocalcaemia and hyperphosphatemia can cause lethargy, fatigue,
bone or joint pain and sudden seizures. Patients may also complain of
nausea, vomiting or constipation and develop cardiac symptoms, including
life threatening ventricular dysrhythmias.

Treatment focuses on increasing Ca and decreasing phosphorus. Give IV

calcium gluconate or calcium chloride.
Aluminium hydroxide gels that bind to phosphorus are given to increase
elimination through the bowel.
Patients with normal renal function may also receive the diuretic
acetazolamide to promote renal excretion of phosphorus.
When necessary, haemodialysis is used to quickly correct
hyperphosphatemia.
In case of hypercalcemia and hypophosphatemia, the cause of
hypercalcemia include excessive use of dietary supplements containing
calcium and vitamin D, which increase calcium reabsorption. A primary
cause of low serum phosphorus is chronic alcoholism.

Symptoms of hypercalcemia and hypophosphatemia include lethargy,

fatigue, changes in mental status, anorexia, nausea and constipation.
Patients may also complain bone pain, pain associated with stones formed
by excess calcium in the kidneys. Cardiovascular effects include
hypertension and conduction abnormalities that can progress to cardiac
arrest.

IV saline infusions and diuretics are given to lower serum calcium through
renal excretion. Steroids may also be administered to decrease intestinal
reabsorption of calcium and mithramycin is given to stimulate calcium
deposits in bones.
Phosphorus replacements can be given orally or by IV infusion.
5. Chlorides and bicarbonates.
Are the major extracellular anions. While both play an important role in
maintaining acid-base balance, bicarbonate is by far the start of the show.
Bicarbonate is the body’s major buffer system. It helps keep the ratio
between acids and bases in a tight range that’s numerically expressed as the
pH.

A rise in pH above normal (alkalosis) or below normal (acidosis) causes

cellular damage and can eventually lead to a patient’s death.

Chloride exists in an inverse relationship with bicarbonate. When there is

too little chloride, bicarbonate builds up and metabolic alkalosis results and
too much chloride, is a bit more complicated- acidotic and dehydration.
One way to determine the cause of acid-base imbalance is by calculating the
anion gap.
Anion gap= Sodium – (Chloride + Bicarbonate)

The normal AG range is 8-12.5

A high anion gap (>12) is an indicative of lactic acidosis in patients with

shock. A normal anion gap with elevated chloride levels should also rise a
flag for acidosis.

A low anion gap indicates alkalosis and hypochloremia from vomiting or

diuretic therapy. Other causes- Cushing’s syndrome, massive blood
transfusions, over-administration of IV fluids containing bicarbonates and
excessive intake of antacids containing sodium bicarbonate can also cause
bicarbonate levels to rise.
Electrolyte imbalance is one possible cause of abnormal heart rhythms or
arrhythmias. The electrical system of the heart depends on the right
amount of electrolytes to function in rhythm, force and rate to provide
adequate blood supply throughout the body.

Congestive heart failure treatment may include medicines to increase the

heart functions such as vasodilators, inotropics, beta-blockers and
angiotensin II receptor blockers.
Cardiac metabolic adaptation in response to hypoxia and Anoxia

Since a constant supply of oxygen is essential to sustain life, body have evolved
multiple defence mechanisms to ensure maintenance of the delicate balance
between oxygen supply and demand.
However, this homeostatic balance is perturbed in response to a severe
impairment of oxygen supply, thereby activating maladaptive signalling
cascades that results in cardiac damage.

Exposure to chronic hypoxia may induce cardio-protective properties. The

hypoxia triggers regulatory pathways that mediate long term cardiac metabolic
remodelling, particularly at the transcriptional levels.

Exposure to hypoxia triggers

a) Oxygen sensitive transcriptional modulators that induce a switch to
increased carbohydrate metabolism.
b) Enhanced mitochondrial respiratory capacity to sustain and increase
efficiency of mitochondrial energy production.
These compensatory protective mechanisms preserve contractile function
despite hypoxia.
Hypoxia is the result of an imbalance between oxygen supply and demand.
Tissue hypoxia is caused by interrupted coronary blood flow and a reduction
in arterial blood oxygen partial pressure.
It is a major contributor to cardiac pathophysiology, including MI.

Chronic O2 lack leads to increased pulmonary constriction that may result in

pulmonary hypertension, increased afterload on the right ventricle that may
eventually lead to heart failure.

Adaptive cardiac metabolic remodelling ie. Switching to greater

carbohydrate versus fatty acid utilization, is proposed to play a key role in
hypoxia mediated cardio-protection.

Fatty acids serve as the major fuel substrate for the normal, fasting adult
hart providing approx. 60-80% of its energy requirements. The rest of the
heart’s ATP is derived from glucose and lactate in nearly equal proportions.
After uptake, glucose is rapidly phosphorylated to glucose-6-p. The latter
has several metabolic fates including its complete oxidation to acetyl-coA
and CO2 via Kreb’s cycle.
Moreover, the partial breakdown of glucose to pyruvate generate ATP
without any O2 requirement while under certain conditions (in hypoxia)
pyruvate may be converted to lactate.

Cardiac fuel substrate utilization is dynamic and may be altered according to

the prevailing physiological or pathophysiological milieu.

Increased glucose utilization in response to hypoxia may occur since it is a

more oxygen efficient fuel substrate to generate ATP compared with fatty
acids.
The regulatory mechanisms directing cardiac metabolic remodelling in
response to hypoxia are complex and the subject of on going research work.
Latest studies has found that cardiac cells augment glycolytic capacity in
response to hypoxia by increasing the expression of genes regulating
glucose uptake and glycolysis.

Also transcript levels of pyruvate dehydrogenase kinase 4, an inhibitor of

pyruvate dehydrogenase and glucose oxidation was reduced while the
expression of the cardiac enriched glucose transporters (GLUT1 and GLUT4)
was not altered. Reduced enzyme activity of beta-hydroxy-acyl-coA
dehydrogenase (of beta-oxidation) was found in response to hypoxia.

It is proposed that ROS relay extracellular signals to the transcriptional

machinery to initiate adaptive cardiac metabolic remodelling. HIF-1alpha
heterodimerizes with HIF-1beta and subsequently binds to hypoxia –
response elements (HRE) within the promoter region to increase expression
of glucose metabolic gene.
Oxygen sensing
Organisms require a sophisticated O2 sensing mechanism to rapidly respond
to alterations in PO2, thereby maintaining intracellular O2 homeostasis.

O2 sensor should meet several requirements, including

a) The ability to sense O2 concentrations
b) Initiating intracellular signalling cascades in response to altered PO2.
c) The capacity to deal with the differing PO2 thresholds for various
organs.
Candidate O2 sensor system in the heart include the NADPH oxidase
enzyme family and components of the mitochondrial ETC (Complex-I, III and
IV).

O2 sensors usually relay alteration in PO2 to the cellular machinary via

intracellular signalling intermediates.

ROS, play a pivotal role in O2 sensing by acting as secondary mesengers,

modulating signalling pathways.
Both NADPH oxidase and the mitochondrial ETC complexes I and III can
produce ROS, thus identifying a potential mechanism whereby an
intracellular response may be initiated to counteract PO2 perturbations.

Thus higher ROS production during hypoxia exposure may play an important
role in transmitting PO2 changes to the cellular apparatus, thereby adapting
responses.

The ROS generated will activate Ca-calmodulin dependent protein kinase to

increase expression of peroxisome proliferator- activated, receptor gamma
co-activator 1-alpha (PGC-1-alpha), a master regulator in mitochondrial
biosynthesis.

This will enhance mitochondrial energy production and attenuate cell death
in the heart when challenged by severe O2 deprivation.
In anoxic stage, lipid accumulation is stimulated that have proven injurious
to subsequent mitochondrial function in cardiac tissues.
When blood flow to a region of cardiac muscle is temporarily or
permanently interrupted, the anoxia that develops and the failure of blood-
flow to transport nutrients and toxic waste to and from the affected area
cause pathological changes, which lead ultimately to lethal cellular injury.

The formation of dense amorphous material in cells has been described and
it has been suggested that these contain lipid and protein.
Lipid accumulation can be observed in anoxic tissues at the periphery of
developing infarcts and is associated with progression of necrosis.