Department of Biochemistry.

Faculty of Medicine, UNHAS

Rosdiana Natzir.

Objectives
Cardiovascular system (Heart)
Anatomical and physiology orientation
Etc
Biochemical Cardiac markers****
Thrombus formation

Course Guideline
Outline of labs
Texts required
Attendance
Grading policy

Learning objectives:
To understand the role of cardium

biomarkers.
To know the role of enzymatic and
non enzymatic cardium biomarkers.
To understand when the cardium
biomarkers were increasing in the
blood level.

Functions of
Cardiovascular system
system circulates blood throughout

body :
transports energy substrates (glucose,

FA, ect.), electrolytes and hormones to

tissues
removes waste products
lactate, CO2 & H2O
thermoregulation

Functions of the Heart

Generating blood pressure
Routing blood: separates pulmonary

and systemic circulations

Ensuring one-way blood flow: valves
Regulating blood supply
Changes in contraction rate and force

match blood delivery to changing

metabolic needs

Example case examination

lab :

Hb
WBC
Sodium
Potassium
Chloride
Urea
Creatinine
CreatineKinase
Troponin I

Blood Flow Through the

Heart

The Cardiovascular System

(cont.)
Blood :
Complex mixture of

cells, water, and

various proteins and
sugars.
Fifty-five percent is
plasma (liquid).
Forty-five percent is
solid.

The Cardiovascular
System (cont.)
Blood (cont.)
Hematocrit measurement of
percentage of red blood cells.
Leukocytes 5 types of white blood
cells protect against disease.

Basophils.
Eosinophils.
Neutrophils.
Lymphocytes.
Monocytes.

Contractility
and
Norepinephrine
Sympathetic

stimulation
releases
norepinephrin
e and initiates
a cyclic AMP
secondmessenger
system

Figure 18.22

Chemical Regulation of
the Heart
The hormones epinephrine and thyroxine

increase heart rate

Intra- and extracellular ion
concentrations must be maintained for
normal heart function

Myocardium - morphology
>

Intercalated disks = the fibers are

connected to each

other in Z lines

Provide strong union between fibers

> Actin, myosin, tropomyosin, troponin

> Large amount of mitochondrias in tight

contact with

fibrils

Myocardium metabolism
Abundant blood supply, numerous

mitochondria, high content of myoglobin (a

muscle pigment) as a storage of O2
Metabolism mostly aerobic, only about 1%

anaerobic (during hypoxia possible up to 10%

anaerobic, if more --> not enough energy for
contractions)
Utilization of substrates depending on the

nutrition 60% fats (mostly FA), 35%

carbohydrates, 5% ketones and AAs

* The Sarcoplasma of muscle cells contains :

ATP; phosphocreatine & glycolytic enzymes.
* the mass of a muscle is made of 75% water and >
20% protein ( actin and myosin ).

Carnitine-palmytoyl transferase -1; malonyl-CoA decarboxylase: acetyl CoA

carboxylase-2; AMP-activated protein kinase.

CARDIAC ENZYMES
OTHER CARDIAC MARKERS

OTHER CARDIAC MARKERS

B NATRIURETIC PEPTIDE (BNP)
N-TERMINAL PRO BNP

PRE-PRO BNP

BNP + NT PRO-BNP

Medical tests that are often referred

to as cardiac markers include:
cardiac troponin (the most sensitive and specific

test for myocardial damage)

creatine kinase (CK, phosphocreatine kinase or
creatine phosphokinase)
Aspartate transaminase (AST,

Glutamic Oxaloacetic Transaminase (GOT/SGOT)

or aspartate aminotransferase (ASAT))
lactate dehydrogenase (LDH)
Myoglobin (Mb) has low specificity for
myocardial infarction and is used less than the
other markers.

 Depending on the marker, it can take between 2

to 24 hours for the level to increase in the blood.

Cardiac markers are therefore not useful in

diagnosing a myocardial infarction in the acute

phase.

The clinical presentation and results from an

ECG are more appropriate in the acute situation.

Troponin
Troponin is a complex of three proteins that is

integral to muscle contraction in skeletal and

cardiac muscle, but not smooth muscle.

Troponin is attached to the protein tropomyosin

and lies within between actin filaments in muscle
tissue.

In a relaxed muscle, tropomyosin blocks the

attachment site for the myosin crossbridge, thus

preventing contraction.

When the muscle cell is stimulated to contract by an

action potential, calcium channels open in the
sarcoplasmic reticulum and release calcium into the
sarcoplasm.

Some of this calcium attaches to troponin, causing a

conformational change that moves tropomyosin out of the

way so that the cross bridges can attach to actin and
produce muscle contraction.

The main difference is that the TnC subunit of troponin in

skeletal muscle has 4 calcium ion binding sites, whereas in

cardiac muscle there are only 3 Ca.

The action potential of a

cardiac cell

The heart muscle cell

contraction

Functional characteristics
Role of troponins :
Both cardiac and skeletal muscles are controlled by

changes in the intracellular calcium concentration

When calcium rises, the muscles contract, and

when calcium falls the muscles relax.

Troponin is a component of thin filaments (along with

actin and tropomyosin), and is the protein to which

calcium binds to accomplish this regulation.

Troponin has three subunits, TnC, TnI, and TnT.

When calcium is bound to specific sites on TnC,
tropomyosin rolls out of the way of the actin filament
active sites,

In the absence of calcium, tropomyosin interferes

with this action of myosin, and therefore muscles
remain relaxed.

Role of troponins :
Troponin I has also been shown to inhibit angiogenesis in vivo

and in vitro

Individual subunits serve different functions:

Troponin C > binds to calcium ions to produce a

conformational change in TnI

Troponin T.> binds to tropomyosin, interlocking them to form

a troponin-tropomyosin complex

Troponin I .> binds to actin in thin myofilaments to hold the

troponin-tropomyosin complex in place

Diagnostic

use :

Certain subtypes of troponin (cardiac troponin I

and T) are very sensitive and specific indicators

of damage to the heart muscle (myocardium).

They are measured in the blood to differentiate

between unstable angina and myocardial infarction

(heart attack) in patients with chest pain.

A patient who had suffered from a myocardial

infarction would have an area of damaged heart

muscle and so would have elevated cardiac troponin
levels in the blood.

 It is important to note that cardiac troponins

are a marker of all heart muscle damage,

not just myocardial infarction.

Other conditions that directly or indirectly lead

to heart muscle damage can also therefore

increase troponin levels:
Cardiac:
Cardiac amyloidosis
Cardiac contusion
Cardiac surgery and heart transplant
Defibrillation

Closure of atrial septal defects

Coronary vasospasm
Dilated cardiomyopathy
Heart failure
Hypertrophic cardiomyopathy
Myocarditis
Percutaneous coronary intervention
Radiofrequency ablation
Supraventricular tachycardia

Detection of cardiac troponin

Cardiac troponin T and I are

measured by immunoassay
methods.

Creatine kinase
Creatine kinase (CK), also known as phosphocreatine

kinase or creatine phosphokinase (CPK) is an enzyme

expressed by various tissue types.

It catalyses the conversion of creatine to phosphocreatine,

consuming adenosine triphosphate (ATP) and generating

adenosine diphosphate (ADP).

In tissues that consume ATP rapidly, especially

skeletal muscle, brain and smooth muscle, phosphocreatine

serves as an energy reservoir for the rapid regeneration of
ATP.

Thus Creatine Kinase is an important enzyme in such tissues.

Clinically, creatine kinase is assayed in blood tests as a marker of

myocardial infarction (heart attack), rhabdomyolysis (severe
muscle breakdown), muscular dystrophy and in
acute renal failure.

Types :
In most of the cell, the CK enzyme consists of two subunits,

which can be either :

B (brain type) or
M (muscle type).

There are, therefore, three different isoenzymes:

CK-MM,
CK-BB
CK-MB.

 The genes for these subunits are located on different

chromosomes: B on 14q32 and M on 19q13.

In addition to those, there are two mitochondrial

creatine kinases, the ubiquitous and sarcomeric form.

CK-BB occurs mainly in tissues, and its levels do

rarely have any significance in bloodstream.

Skeletal muscle expresses CK-MM (98%) and low

levels of CK-MB (1%).

 The myocardium (heart muscle), in

contrast, expresses CK-MM at 70% and CKMB at 25-30%. CK-BB is expressed in all
tissues at low levels and has little clinical
relevance.
The mitochondrial creatine kinase (CKm),

which produces ATP from ADP by converting

creatine phosphate to creatine, is present
in the mitochondrial intermembrane
space.

Apart from the mitochondrial form, there are three

forms present in the cytosol
CKa : (in times of acute need, produces ATP in the
cytosol at the cost of creatine phosphate),
CKc : (maintains critical concentration of creatine
and creatine phosphate in the cytosol by coupling
their phosphorylation and dephosphorylation
respectively with ATP and ADP) and
CKg : (which couples direct phosphorylation of
creatine to the glycolytic pathway.

creatine

Creatin kinase, muscle

Aspartate transaminase
Aspartate transaminase (AST) also

called serum glutamic oxaloacetic

transaminase (SGOT) or aspartate
aminotransferase (ASAT/AAT)
is similar to alanine transaminase (ALT)

in that it is another enzyme associated

with liver parenchymal cells.

Function

AST

It facilitates the conversion of aspartate and

alpha-ketoglutarate to oxaloacetate and glutamate

Isozymes

Two isoenzymes are present in humans. They

have high similarity.
GOT1, : the cytosolic isoenzyme derives mainly
from red blood cells and heart.
GOT2, : the mitochondrial isoenzyme is
predominantly present in liver

Aspartate aminotransferase.
Aspartate aminotransferase from Escherichia coli
bound with cofactor pyridoxal 5-phosphate (PDB 1AAM)

Lactate dehydrogenase
Lactate dehydrogenase (LDH) is an

enzyme present in a wide variety of

organisms, including plants and animals.
Reactions:

It catalyses the interconversion of

pyruvate and lactate with concomitant
interconversion of NADH and NAD+.
.

At high concentrations of lactate, the enzyme exhibits

feedback inhibition and the rate of conversion of pyruvate
to lactate is decreased

Catalytic function of LDH

Enzyme isoforms :
LDH-1 (4H) - in the heart
LDH-2 (3H1M) - in the reticuloendothelial system
LDH-3 (2H2M) - in the lungs
LDH-4 (1H3M) - in the kidneys
LDH-5 (4M) - in the liver and striated muscle

 The major isozymes of skeletal muscle and

liver, M4, has four muscle (M) subunits;

while H (heart)4 is the main isozymes for
heart muscle in most species, containing 4 H
subunits.

The other variants contain both types of

subunits.

Usually LDH-2 is the predominant form in the

serum.

 A LDH-1 level higher than the LDH-2 level ,

suggests myocardial infarction (damage to

heart tissues releases heart LDH, which is rich
in LDH-1, into the bloodstream).
The use of this phenomenon to diagnose

infarction has been largely superseded by

the use of Troponin I or T measurement.

Myoglobin
Myoglobin is a single-chain globular

protein of 153 amino acids,

containing a heme (iron-containing

porphyrin) prosthetic group in the

center around which the remaining
apoprotein folds.

It has a molecular weight of 16,700

daltons, and is the primary oxygencarrying pigment of muscle tissues.

Model of helical
domains in myoglobin

Molecular Function:
oxygen transporter activity
iron ion binding
oxygen binding
heme binding
metal ion binding

Biological Process:
response to hypoxia
transport
oxygen transport
enucleate erythrocyte differentiation

Role in disease
Myoglobin is a sensitive marker for muscle injury,

making it a potential marker for heart attack in

patients with chest pain.

CK-MB and TnT is used in combination with ECG,

and the clinical signs to diagnose AMI

Myoglobin is released from damaged muscle tissue (

rhabdomyolysis), which has very high

concentrations of myoglobin.

The released myoglobin is filtered by the kidneys but

is toxic to the renal tubular epithelium and so may

cause acute renal failure.

Structure and bonding

Myoglobin contains a porphyrin ring with an

iron center. There is a proximal histidine

group attached directly to the iron center.

It serves three functions:

to form hydrogen bonds with the dioxygen
moiety, increasing the O2 binding constant

 to prevent the binding of carbon monoxide.

CO binds to heme 23,000 times better than

O2, but only 200 times better in hemoglobin

and myoglobin. Oxygen binds in a bent
fashion, which can fit with the distal histidine.

to prevent irreversible dimerization of the

with another species

Glycogen phosphorylase
isoenzyme

is an isoenzyme of glycogen phosphorylase. This

isoform of the enzyme exists in cardiac (heart) and
brain tissue.

The enzyme is one of the "new cardiac markers"

which are discussed to improve early diagnosis in

acute coronary syndrome.
A rapid rise in blood levels can be seen in
myocardial infarction and unstable angina.
Other enzymes related to glycogen phosphorylase

are abbreviated as (liver) and (muscle).

Biochemical analysis
A basic metabolic panel measures sodium, potassium,

chloride, bicarbonate, blood urea nitrogen (BUN),

magnesium, creatinine, and glucose. It also sometimes
includes calcium.

Some blood tests, such as measuring glucose, cholesterol,

screening require fasting (or no food consumption) eight

to twelve hours prior to the blood test.

Blood gas analysis of arterial blood is primarily used to

monitor carbon dioxide and oxygen levels related to
pulmonary function. But, it is also used to measure blood pH
and bicarbonate levels for certain metabolic conditions.

the glucose tolerance test involves repeated

testing to determine the rate at which glucose
is processed by the body.

The diagnosis of myocardial infarction requires

two out of three components (history, ECG,

and enzymes).

Normal ranges
Test

Low

High

UnitComments

Sodium (Na)

136

145

mmol/L

Potassium (K)

3.5

4.5

mmol/L

Urea
urea nitrogen

Urea

Creatinine male

62

115

mol/L

Creatinine female

53

97

mol/L

Creatinine male

0.7

1.3

mg/dL

Creatinine female

0.6

1.1

mg/dL

Glucose (fasting)
3.9
glycosylated hemoglobin

5.8

mmol/L See also

Glucose (fasting)

105

mg/dL

2.5

70

6.4

mmol/L

18

mg/dL

BUN - blood

Thrombus
formation

Functions
There are six functions:
1. Transportation
O2 lungs cells
CO2 cells lungs
Nutrients GI cells
waste from cells kidneys

Functions

2. Defense

WBC disease
blood proteins antibodies

Function
s

3. Temperature regulation - absorbs

and distribute heat throughout body
and skin
4. Prevents loss blood clots
5. Hormone movement endocrine
gland cells
6. Regulates pH through buffers

Imbalances (Disorders of
Hemostasis)
Thrombus clot that develops and

persists in an unbroken vessel which

can lead to thrombosis where
tissue/organ die because of obstruction
(coronary thrombosis)
Embolism when small vessel is
obstructed from embolus .
Thrombocytopenia number of platelet
is deficient in blood

 Normalnya darah yg mengalir tetap cair

ok keseimbangan kompleks tertentu

tetap ada.
Bila terganggu -----> trombosis.
Melekatnya trombosis pada permukaan
endotel pembuluh darah atau jantung
----> darah mengalir ----> trombosit
tertimbun jika aliran darah cepat ---->
lepas dari dinding pembuluh darah
kmd diganti oleh trombosit lain.

 Bila trombosit rusak ---->

tromboplastin dikeluarkan ---->

merangsang pembentukan beku
darah dan mengubah protrombin
----> trombin dan bereaksi
dengan fibrinogen -----> fibrin.
Fibrin seperti jala mudah mengikat
RBC dan WBC .
Bentuk trombus khas .

Menurut lamanya terjadi

trombus :
Fresh trombus
Old trombus

Macam2 trombus :
Occlusive trombus : lumen

pembuluh darah tersumbat.

Propagating trombus : massa
sepanjang pembuluh darah
terbendung dan merupakan
perpanjangan trombus.
Saddle/riding trombus : memanjang
dan masuk kedalam cabang
pembuluh darah.

 Mural/parietal trombus : trombus yg

melekat dan lepas, bagian yg lepas

seolah-olah berenang dalam darah
tanpa oklusi pembuluh.
Pedinculated trombus : trombus
mural yang bertangkai panjang.
Ball trombus : pedinculated trombus
yg lepas memasuki aliran darah
besar.

Apa yang menyebabkan

trombus
Menurut Virchow bhw trombus

terbentuk oleh 3 faktor :

Perubahan pada permukaan endotel
endotel pembuluh darah.
Perubahan pada aliran darah.
Perubahan pada konstitusi darah.

Example assessment :

Myocard
infarction
12 lead ECG (ST elevation)
Pathologic Q wave
Inverted T wave

CPK-MB elevates 2-4 hours

CPK enzymes, cardiac specific fraction 12-18 hours
SGOT 24-36 hours
LDH peaks 48-72 hours

CAUSES:
EMBOLISM
Formation of plaque
Along the coronary
lumen
Decreased O2
In areas of the
heart
Glycogen stores
depleted

Lactid acid
accumulates

Tissue ischemia

Tissue necrosis

20 minutes:
It takes
For a necrotic
cardiac
Tissue to die

Cell death

Diagnostics:
- Serial Electrocardiogram (ECG)
-

echocardiogram: ventricular function

- serum laboratory studies:

a. cardiac isoenzyme: Creatinine
phosphokinase,
b. Myoglobin and troponin levels are elevated.
c. increase in white blood cells count
d. Increase in B-type Natriuretic peptide.

Clinical Manifestation
Myocardial Infarction
Lab Diagnostics
Cardiac Protein Troponin T
More sensitive than CK
Elevates 3 hr peak 24-48 hrs; normal 5-14 days

Cardiac Enzyme Creatine kinase (CK-MB)

Released when cardiac cells die
Elevates 3 hrs peak 12-24 hrs; normal 2-3 days

Cardiac Marker - Myoglobin

First to elevate
Lacks cardiac specificity
Normal range within 24 hours

Serum Cardiac Markers

after MI

Glucose utilization in the

cardiomyocyte.

Glucose uptake into

cardiomyocyte occurs
through GLUT1 and
GLUT4 transporters

 Once inside, glucose is broken down through glycolysis,

that convert glucose into pyruvate.
Phosphofructokinase-1 (PFK1), the enzyme that catalyze
the generation of fructose 1,6-bisphosphate from fructose 6 phosphate, is a rate-limiting enzyme controlling glycolysis.
PFK1 is activated by 2,6-bisphosphate, which is formed
from fructose 6-phosphate catalyzed by PFK-2.

 After glycolysis, the pyruvate generated is

transported into mitochondria and

decarboxylated to acetyl-CoA through
pyruvate dehydrogenase (PDH), a
multienzyme complex.
PDH is phosphorylated and inactivated by

pyruvate dehydrogenase kinase (PDK).

Acetyl-CoA then enters tricarboxylic acid cycle
(citric acid cycle) and is eventually broken
down to H2O and CO2 for ATP generation.

AMP-activated protein kinase control of

energy metabolism in the ischemic heart
AMPK the ischemic heart
Myocardial ischemia :
produces an energy-deficient state in heart
muscle, which if not corrected can lead to
cardiomyocyte death.

AMP-activated protein kinase (AMPK) is a

key kinase that can increase energy
production in the ischemic heart.

During ischemia a rapid activation of AMPK

occurs, resulting in an activation of both
myocardial glucose uptake and glycolysis,
as well as an increase in fatty acid
oxidation.

Eeukaryote elongation factor-2

Fphosphofructokinase 2

Control of glycolysis and

protein synthesis by
ischaemia in heart

Metabolic targets of
AMPK

For your attention

Thank you very much