SERUM HEART TYPE FATTY ACID BINDING
PROTEIN (HFABP) AS AN EARLY MARKER OF
MYOCARDIAL ISCHEMIA
Dissertation Submitted for
M.D DEGREE BRANCH - XIII
[BIO CHEMISTRY]
DEPARTMENT OF BIOCHEMISTRY
K.A.P.V GOVT. MEDICAL COLLEGE,
TRICHY-1.
THE TAMILNADU DR.M.G.R MEDICAL UNIVERSITY,
CHENNAI -32
APRIL – 2017
CERTIFICATE
This is to certify that dissertation titled “SERUM HEART TYPE FATTY
ACID BINDING PROTEIN (HFABP) AS AN EARLY MARKER OF
MYOCARDIAL ISCHEMIA” is a bonafide work done by
Dr.P.DEEPALAKSHMI under my guidance and supervision in the Department
of Biochemistry, K.A.P.V Govt. Medical College, Trichy-1, during her post
graduate course from 2014 to 2017.
Prof. Dr. S. MARY LILLY.M.D,(Path).,
THE DEAN,
K.A.P.V Govt. Medical College,
Trichy-1.
Dr. K. NIRMALADEVI.M.D.(Bio),D.C.H.,
PROFESSOR AND HOD,
Department of Biochemistry,
K.A.P.V Govt. Medical College,
Trichy - 1.
GUIDE CERTIFICATE
The work done by DR.P.DEEPALAKSHMI on “SERUM HEART
TYPE FATTY ACID BINDING PROTEIN (HFABP) AS AN EARLY
MARKER OF MYOCARDIAL ISCHEMIA” is under my supervision and I
assure that this candidate has followed the rules of the Ethical Committee.
GUIDE : Dr. K. NIRMALADEVI, M.D.(Bio),D.C.H.,
THE PROFESSOR AND HOD,
DEPARTMENT OF BIOCHEMISTRY,
K.A.P.V GOVT. MEDICAL COLLEGE,
TRICHY - 1.
DECLARATION
I, Dr.P.DEEPALAKSHMI hereby solemnly declare that the dissertation
titled “SERUM HEART TYPE FATTY ACID BINDING PROTEIN
(HFABP) AS AN EARLY MARKER OF MYOCARDIAL ISCHEMIA”was
done by me at K.A.P.V Govt. Medical College & MGMGH, Trichy under the
Supervision and Guidance of my Professor and H.O.D Dr. K.NIRMALADEVI,
M.D.(Bio),D.C.H. This dissertation is submitted to Tamil Nadu Dr. M.G.R
Medical University, towards partial fulfillment of requirement for the award of
M.D. Degree (Branch –XIII) in Biochemistry.
Place: Trichy
Date: DR.P.DEEPALAKSHMI
ANTI – PLAGIARISM – ORIGINALITY REPORT
ACKNOWLEDGEMENT
I am thankful to the Almighty who is always. I am extremely grateful
toProf.Dr.S.MARY LILLY, M.D (Path)., The Dean, K.A.P.V.Govt. Medical
College for permitting me to do this dissertation at MGMGH, Trichy. I am
indebted greatly to my Professor and Head of the Department,
Dr. K. NIRMALADEVI, M.D.(Bio),D.C.H., Department of Biochemistry, who
had inspired, encouraged and guided me in every step of this study.
I express my heartiest thanks to Dr.A.ARSHIYA BEGUM M.D.,
Additional Professor of Biochemistry, K.A.P.V.Govt.Medical College. I express
my sincere gratitude to DR.P.KANAGARAJ. M.D., former Professor and H.O.D,
Department of General Medicine and Dr.T.BALASUBRAMAINAN. M.D., D.M
(Cardio)., Associate Professor of Cardiology, MGMGH, for their valuable help.
I also express my heartiest thanks to Dr.R.PANIMATHI,M.D(Bio),
D.C.H., former Associate Professor of Biochemistry, K.A.P.V.Govt. Medical
College for her help and suggestions for performing my study. I would like to
thank DR.P.SELVAM, M.D., Assistant Professor, Department of Community
Medicine, K.A.P.V.Govt. Medical College, for his help in statistical analysis and
successful completion of my dissertation.
I would like to thank all the Assistant Professors, Department of
Biochemistry, K.A.P.V.Govt. Medical College for their help and support during
my study.
I owe my thanks to my co-post graduates for their support during the
study. I would like to acknowledge the assistance rendered by the Technical staffs
who helped me to perform the study.
I am grateful to all my patients and volunteers who participated in this
study. I owe my special thanks to my family members for their moral support in
conducting the study.
CONTENTS
S No PARTICULARS PAGE NO.
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 3
3 AIMS AND OBJECTIVES 28
4 MATERIALS AND METHODS 29
5 RESULTS AND STATISTICAL ANALYSIS 56
6 DISCUSSION 83
7 CONCLUSION 86
8 LIMITATIONS OF THE STUDY 87
9 SCOPE FOR FUTURE STUDY 88
10 ANNEXURES
1. Bibliography 89
2. Proforma
3. Consent Form
ABBREVIATIONS
ACS ACUTE CORONARY SYNDROME
AMI ACUTE MYOCARDIAL INFARCTION
AST ASPARTATE TRANSAMINASE
BMI BODY MASS INDEX
CAD CORONARY ARTERY DISEASE
CK-MB CREATININE KINASE- MB ISOFORM
DM DIABETES MELLITUS
ECG ELECTROCARDIOGRAM
FABP FATTY ACID BINDING PROTEIN
HDL HIGH DENSITY LIPOPROTEIN
HFABP HEART TYPE FATTY ACID BINDING PROTEIN
HT HYPERTENSION
IHD ISCHEMIC HEART DISEASE
LDL LOW DENSITY LIPOPROTEIN
MI MYOCARDIAL INFARCTION
NSTEMI NON ST ELEVATED MYOCARDIAL INFARCTION
PAI-1 PLASMINOGEN ACTIVATOR INHIBITOR-1
PPAR PEROXISOME PROLIFERATOR ACTIVATED RECEPTORS
STEMI ST ELEVATED MYOCARDIAL INFARCTION
TGL TRIGLYCERIDE
UA UNSTABLE ANGINA
WHO WORLD HEALTH ORGANIZATION
1
INTRODUCTION
Cardiovascular Disease is a major health problem across the world. By
the year 2020, one in every three deaths will be due to CVD.1 Eighty
Percentage of total deaths in developing countries are due to cardiovascular
disease.2 Ischemic Heart Disease is the most common cause of cardiovascular
morbidity and mortality.
IHD occurs due to the imbalance between the oxygen supply and
oxygen demand of the myocardium. The manifestations of IHD are Angina
Pectoris, Unstable Angina Pectoris, Myocardial Infarction, Heart failure and
sudden cardiac death.3 Patients may have transient underlying pathology before
the signs and symptoms of AMI become apparent.
Early detection of AMI at the ischemic stage is important to prevent
morbidity and mortality. The diagnosis of AMI is based on history, clinical
examination, electrocardiogram and biochemical markers. ECG is not
sufficient to diagnose the IHD since ST segment changes can also be observed
in other conditions.4 Cardiac markers are also important in the diagnosis of
AMI. Myoglobin, CK-MB, Cardiac Troponin- T and Troponin-T are the
currently used biochemical markers for diagnosing IHD.
Detection of AMI in the early hours by these markers is not
satisfactory,5 since these markers tend to elevate only after 6 hours of onset of
injury. Even though myoglobin level increases within 2 hours, the specificity of
2
myoglobin towards the myocardium is low. Recent data show that H-FABP is a
sensitive indicator of ischemia, increases earlier than other cardiac markers.6 It
is also possible to detect the myocardial damage soon as an hour after onset of
injury by H-FABP level. It’s level is elevated early in the ischemic stage.7
Diagnosing AMI at this stage helps in preventing the progression of disease. Its
diagnostic accuracy may be better than other cardiac markers in the early
stages.
This study is done to determine the efficacy of H-FABP and CK-MB in
the early diagnosis of MI patients admitted with complaints of chest pain.
Serum H-FABP level is estimated and correlated with CK-MB and Lipid
profile.
3
REVIEW OF LITERATURE
The concept of cardiovascular continuum is a chain of events connecting
cardiovascular risk factors with progressive development of pathological
changes which include tissue remodelling finally cardiac failure and death.8
Coronary Heart Disease has been defined as impairment of heart function due
to inadequate blood flow compared to its need.
Epidemiology
Epidemiological transition is driven by industrialization, urbanization
and associated life style changes and it is taking place in every part of the
world. India is experiencing an alarming increase in heart disease. IHD is
growing among low income groups. Population subgroups that appear to be
particularly affected are men in south Asian countries especially India and
Middle East.9 While the incidence of coronary artery disease has reduced by
50% in the west, in India, it has doubled in the last 25 years. The presence of
coronary artery disease in the years 1960, 1980, 1990 and 2000 progressively
increased from 2%, 4 to 6%, 9.5% and 10 to 15% respectively. In the rural side,
CAD prevalence increased 2 fold from 2 to 4 % and in urban India it has
increased 3 fold from 3.45% to 9.45%.10
WHO has predicted from the year 2000 to 2020, DALYS (Disability
Adjusted Life Years Lost) from CHD in India shall double in men and women
from 7.7 and 5.5 million respectively.11
At the beginning of 21st century,
4
cardiovascular disease accounted for nearly half of all the deaths in the
developed and 25% in the developing world.12
Risk Factors
The epidemiology of global heart disease reflects inequalities in risk
factor prevalence.
Non modifiable risk factors or Immutable risk factors:13
1. Age
2. Sex
3. Ethnicity
4. Family history
5. Genetic factors
6. Type A Personality
Modifiable risk factors:
1. Hypertension
2. Diabetes mellitus
3. Elevated total Cholesterol and LDL
4. Tobacco chewing
5. Alcohol
Life style risk factors:
1. Obesity
2. Physical inactivity
3. Atherogenic diet
5
Emerging or non-traditional risk factors:
1. Pro-thrombotic factors- e.g.: fibrinogen, PAI
2. Pro inflammatory factors
3. Impaired fasting Glucose
4. Subclinical atherosclerosis
5. Elevated Homocysteine
Casual risk factors:
1. Lipoprotein(a)
2. Apolipoprotein
6
CORONARY RISK FACTOR FOR ASIAN INDIANS14
FIXED MODIFIABLE
NON- LIPID MODIFIABLE LIPID
MODIFIABLE
LIPOPROTEIN
RATIO
Male age
>35years
Hypertension Total cholesterol>
150 mg/dl
TC/HDL > 4.5
Smoking LDL/HDL >3.5
DM/Insulin resistance
Syndrome
TGL > 150 mg/dl Apo A/Apo B<1.2
Female
age>45years
LDL > 100 mg/dl
BMI > 23 HDL < 40mg/dl in
Homocysteine>
10 mmol / L
males
Family
history of
premature
CHD<55rs
HDL < 50mg/dl in
High PAI >8560
pg/mL
females
Apo A lipoprotein<
100mg/dl
Immutable Risk Factor
Age:
The peak period for coronary heart disease is between 40 to 60 years.15
But it is often detectable in young men between 20 to 30 years of age.16
Sex:
Incidence of IHD is more common in men when compared to women.
Due to estrogenic effects, occurrence of IHD is low in premenopausal women.
After menopause the risk increases up to the level of men.17
Oral intake of
contraceptive pills in premenopausal women increases the risk of IHD by
7
accelerating Hypertension. Oral contraceptive pills have dose related effects on
Blood Pressure. This effect is due to Oestrogen induced increase in renin
substrate.18
Family History:
First degree relatives are at more risk. IHD in males less than 45 years,
females less than 55 years increases the risk of premature death.19
Genetic History:
Genetic predisposition to coronary heart disease is polygenic.
Psychosocial Factor:
Stress, loneliness and social deprivation have major influences on
cardiac disease. Type A behaviour includes time conscious, in-secureness,
being impatient, incapable of relaxation, competitive, aggressive have double
the risk of CAD when compared to normal healthy individuals.20
Modifiable Risk Factors
Hypertension:
Hypertension is an independent risk factor for atherosclerosis. In
persons aged 35 to 65 years, hypertension causes 2 to 4 fold increase of
atherosclerotic events. When BP exceeds 140/90 mmHg, there is 5 fold
increase of CAD.21
8
Diabetes mellitus:
Diabetics are more prone to IHD than non-diabetics by 2 to 3 times.
Endothelial dysfunction and persistently activated thrombogenic pathway and
impaired fibrinolysis in diabetic individuals favour IHD.22,23
Thirty Percentage
to 50% of death in diabetics over the age of 40 years in industrialized countries
are due to DM.24,25
Tobacco chewing:
The risk of CHD is related to number of cigarettes smoked per day.
Smokers- 1 pack/day have 3 to 5 times more death risk than non-smokers.26
1. Nicotine increases the cardiac demand by stimulating the release
of catecholamines.
2. Carbon monoxide induces atherosclerosis and relative
hypokalaemia.
3. Dyslipidaemia- decreased HDL and increased LDL.
4. Nicotine decreases the myocardial oxygen supply by inducing
vasospasm and platelet aggregation.
5. Enhances the coagulability and inflammatory state.
Alcohol intake:
Intake of 75g or more per day becomes an independent risk factor for
IHD.27
9
Lifestyle Risk Factors
Obesity:
Greater the weight gain (BMI >30kg/m2) greater is the risk of
Hypertension, CHD and insulin resistance Diabetes Mellitus. Atherosclerosis is
more common in android type of obesity.28
Physical Inactivity:
Sedentary life style with reduced physical activity leads to an early
development of CHD. Exercise helps in the formation of collateral vessels and
increases the HDL-C.29
Atherogenic Diet:
Fewer intakes of vegetables, fruits and Poly Unsaturated Fatty Acids
(PUFA) increases the risk of CHD. Supplementation of Vitamin E, β Carotene,
Folate and ω3 fatty acids reduces the risk.
Non Traditional Risk Factors
Homocysteine:
Homocystinuria- latest in the series of inborn error of metabolism is
autosomal recessive in nature. It is due to defect in methionine metabolism.
Deficiency of the enzyme Cystathionine Beta Synthase (CBS) leads to
increased homocysteine level in blood.30
10
CBS
Homocysteine + Serine Cystathionine
PLP
Increased homocysteine causes increased platelet adhesiveness and life
threatening intravascular thrombosis which leads to early atherosclerosis and
young Myocardial Infarction.31
Lipoprotein (a)
It is a glycoprotein of structural homology with plasminogen. Higher
levels of lipoprotein (a) compared to other ethnic groups have been recorded in
Asian Indians.32, 33
Prothrombotic Factors
Increased level of fibrinogen increases the risk of CAD.34
Ischemic Heart Disease
The most frequently recognized cause of myocardial ischemia is
atherosclerotic disease of epicardial coronary artery sufficient to cause a
regional reduction in myocardial blood flow and inadequate perfusion of the
myocardium supplied by involved coronary artery. Atherosclerosis either
occludes or narrows the vessel lumen primarily or may secondarily induce a
coronary thrombus. IHD can manifest as chronic stable angina or CAD. CAD
includes STEMI, NSTEMI and unstable angina pectoris.
11
Etiology of IHD
Main cause of ischemia is reduction in blood supply and increase in
oxygen demand.35
Causes of reduced blood flow:
1. Atherosclerosis
2. Coronary spasm
3. Arterial thrombi
4. Coronary emboli
5. Ostial narrowing due to arteritis.13
Increased oxygen demand:
1. Aortic stenosis which causes severe left ventricular hypertrophy.
2. Carboxy haemoglobin which reduces oxygen carrying capacity.
Pathogenesis of Atherosclerosis
Atherosclerosis is not a single disease entity. The process of
atherosclerosis begins in childhood with the development of flat lipid rich
lesions caused by fatty streaks. Atherosclerosis is a chronic inflammatory
disorder of the intima of large and medium sized arteries characterized by
recruitment of monocytes and T-lymphocytes and induction of fibrosis with
smooth muscle cell proliferation and matrix synthesis.36
Modified response to
injury hypothesis explains the initiation and development of atherosclerosis.
12
Modified Response To Injury Hypothesis37
1. Adhesion of monocytes to the endothelium followed by migration into
the intimal layer and transformation into macrophages and foam cells.
2. Lipoproteins, mainly LDL and its oxidized form get accumulated in the
vessel wall.
3. This leads to the formation of fatty streaks which disrupts the
endothelium.
4. Adhesion of platelets.
5. Recruitment of smooth muscle occurs due to the factors released from
activated platelets, macrophages and vascular cell wall.
6. Proliferation of smooth muscle and production of extracellular matrix.
7. Accumulation of lipids occurs both extracellular and intracellular.
13
PIC 1: PATHOGENSIS OF ATHEROSCLEROSIS
Modified lipoprotein triggers a local inflammatory response which leads
to the migration of monocyte derived macrophages and lymphocytes,
converting them to foam cells, mediated by cytokines IL-1 (Interleukin-1) and
TNF (Tumour Necrosis Factor).38,39
Activated leukocytes and vascular wall
cells release growth factor.40,41
Inflammatory markers like CRP-C reactive
protein, Interleukin 6, Myeloperoxidase well correlates with the risk of IHD.42
14
Acute Changes of Plaque
Acute changes of plaque include rupture, fissuring, ulceration which
leads to exposure of thrombosed plaque constituents or underlying sub
endothelial basement membrane. Intra plaque haemorrhage leads to expansion
of plaque volume and causes obstruction.43
Pic 2: Acute Changes of Palque
Thrombotic Occlusion
Rupture of thin fibrous cap causes thrombosis which leads to unstable
angina.44
The site of plaque rupture form the nidus for thrombi by allowing
blood coagulant factors to contact thrombogenic collagen found in the arterial
extracellular matrix and tissue factor produced by macrophage derived foam
cells in the lipid core of the lesion.
15
Pathophysiology of IHD:
The extend and irreversibility of myocardial damage depend on
1. The metabolic needs of the under perfused tissue.
2. Existing collateral vessels
3. Location, severity, duration and rate of development of arterial
occlusion.45
Pic 3: Pathogensis of Coronary Artery Disease
16
Stable Angina
Stable angina is due to fixed atheromatous plaque which causes stenosis
of one or more coronary arteries which causes reduction in blood flow.46
Seventy Percentage to 75% or more obstruction of the vessel lumen causes
symptomatic angina. It is characterized by exertional chest pain and is relieved
by rest.
ECG findings
May be normal but sometimes show ST segment depression on
Treadmill ECG.
Acute Coronary Syndrome
Spectrum of disease that encompasses ischemia with minimal
myocardial damage.47
Myocardial damage includes myocardial infarction and
unstable angina. The precipitating event is alteration in the structure of plaque
which leads to the development of thrombus on the ulcerated or cracked
atherosclerotic plaque. The episodes of plaque disruption may result in episodic
ischemic symptoms with minimal myocardial damage.48
17
BOX 1: CLASSIFICATION OF ACS
Unstable Angina
This is due to dynamic obstruction of a coronary artery due to plaque
rupture with super imposed thrombosis and spasm.49
It is characterized by new
onset or rapidly worsening angina, angina on minimal exertion or at rest.
ECG Findings
1. ST depression.
2. Transient ST elevation
3. T wave inversion in 30 to 50 % of patients
4. No elevation of cardiac bio markers such as CKMB and Troponin.
18
Myocardial Infarction
This term is applied to myocardial necrosis secondary to an acute
interruption of blood supply. Occlusion of blood flow leads to cardiac myocyte
death via oncotic and apoptotic pathways. This leads to acute exudative
inflammation with neutrophil and monocyte invasion. Tissue repair occurs via
the activation of supportive stromal cells including fibroblast and endothelial
cells. Inflammatory reactions gradually resolve and granulation tissues,
leukocytes, fibroblasts and vascular cells undergo apoptosis. This results in a
cellular scar devoid of contractile function which leads to compensatory
dilatation and remodelling of left ventricles.
ECG Findings of STEMI:
1. ST segment elevation
2. Q wave formation
3. Elevated cardiac biomarkers.
NSTEMI
This is due to partially occluded thrombus forming on a disrupted
atherosclerotic coronary plaque or on eroded coronary artery endothelial cells.
Myonecrosis occurs without elevation of ST segment.50
Cardiac bio markers
CKMB and Troponin will be elevated.
ECG Findings:
1. T wave inversion and ST depression exceeding 2 mm but no ST elevation.
Formation of new and deep T waves (≥0.3mV).
19
Cardiac markers will be elevated in both STEMI and NSTEMI. The
biomarkers commonly used are CK-MB, Troponin I, and Myoglobin. Even
though Myoglobin level increases within 2 hours, its specificity towards
myocyte is very low. CK-MB rises within 4 to 8 hours, peaks at 12 to 24 hours
and returns to normal level by 48 – 72 hours. Troponin tends to elevate 4 to 10
hours after the onset of symptoms, peaks at 12-48 hours and reaches the normal
level by 4 to 10 days. HFABP which is a cardiac specific marker tend to
elevate earlier than other markers.6
Pic 4: Relationship Among Acute Coronary Syndrome, Inflammation,
Ischemia And Chronic Heart Failure with Atherosclerosis
From Update on Cardiac markers by Eileen Carreiro – Volume 37, No.10
October 2006, lab medicine
20
FATTY ACID BINDING PROTEINS (FABPs)
FABPs belong to multigene family. They are highly conserved cytosolic
proteins with a molecular mass of 14 to 15 K Da. One to 5% of all soluble
cytosolic protein is made up of FABPs.51
They are ubiquitously expressed in all
the tissues but differ in stoichiometry, affinity and specificity towards ligand.
They are abundant in tissues with active Fatty Acid metabolism.52
They have
high affinity towards long chain fatty acids and hydrophobic ligands.
Mammalian family includes 9 types of FABPs.
21
Isoforms of Fatty Acid Binding Proteins:53
Approved
Symbol
Approved Name
Previous
Symbols
Synonyms
Chromosome
FABP1
Fatty acid binding protein
1, liver
L-FABP
2p11
FABP2
Fatty acid binding protein
2, intestinal
I-FABP
4q28-q31
FABP3
Fatty acid binding protein3,
muscle and heart(mammary
derived growth inhibitor)
MDGI,
FABP1
H-FABP,
O-FABP
1p33-p32
FABP4
Fatty acid binding protein4,
adipocyte
A-FABP,
8q21.13
FABP5
Fatty acid binding protein5
(psoriasis-associated)
E-FABP, PA-
FABP,
KFABP
8q21.13
FABP6
Fatty acid binding protein
6, ileum.
I-15P,
ILLBP,I-
BAP, ILBP3,
I-BABP,
ILBP,
I-BALB
5q23-q35
FABP7
Fatty acid binding protein
7, brain
B-FABP,
BLBPs
6q22-q23
FABP9
Fatty acid binding protein
9, testis
PERF,
T-FABP,
PERF15
8q21.13
FABP12
Fatty acid binding protein
12
8q21.13
22
Structure
All FABP family members have 20 to 70% sequence homologies. But
they differ in their tertiary structure.54,55
The divergent sequence among family
members confer difference in protein - protein interaction and different ligand
binding properties depending on their cellular location.56
All FABPs exhibit complicated tertiary structures and their 10 anti-
parallel β strands are organized into 2 orthogonal β sheets, which form a
slightly elliptical β barrel with two 8 to 10 residue helixes that links the first 2 β
strands together.57
Ligand binding cavity extends from the helix turn helix
motif which act as a portal for Fatty Acid access and egress.58
The helical N
terminus is involved in the Fatty acid transfer via electrostatic interaction.53
Pic 5: Apo and Holo FABP3
23
Functions
FABPs act as a carrier protein for fatty acid transport and other
lipophilic substances from the cytoplasm to nucleus via nucleus receptors such
as Peroxisome Proliferator Activated Receptors (PPARs).59
Peroxisome Proliferator Activated Receptors (PPARs):
PPARs are nuclear receptors. They are transcription factors, affect many
metabolic processes in response to a variety of fatty acid like ligands.60
It
activates the gene essential for fatty acid oxidation including fatty acid
transporter. This response is triggered when a cell or organism has an increased
demand for energy for fat metabolism. FABPs bind with Fatty acids through
the ligand binding cavity and helps in intracellular uptake. There are three
types of PPARs- α, β, and γ. This α receptor which is present in Liver, Heart,
Kidney, Skeletal muscle and Brown adipose tissue is involved in the
metabolism of fat.61
24
Pic 6: Mechanism of Action of HFABP
25
Heart Type Fatty Acid Binding Protein (HFABP, FABP 3)
HFABP is a small intracellular cytoplasmic protein consists of 132
amino acids. It weighs about 14.5 K Da and is water soluble.62
Molecular genetics
FABP3 gene is located at chromosome 1 p33-p31. The overall genetic
structure of FABPs consist of 4 exons separated by 3 introns. The position of
exons and introns are same in all FABPs but the length of introns varies in
isoforms. The length of intron-1 in FABP3 is 3.4 kb. The distinct distribution
of HFABP is regulated by concise promoter regions within the gene. The 1.2
Kb promoter region is required for tissue specific expression of HFABP in
heart.63
The same gene is also expressed in the mammary gland in a highly
regulated manner, where it was initially named as Mammary Derived Growth
Inhibitor (MDGI). Its function is to arrest growth of mammary epithelial cells
and it is a tumour suppressor for human breast cancer.
26
Pic 7: Heart Type Fatty Acid Binding Protein (HFABP)
Distribution
Among the HFABPs, heart type of FABP is most widely distributed. It is
found in heart, skeletal muscle, smooth muscle, mammary epithelial cells, aorta,
distal tubules of kidney, lung, brain, placenta and ovary.64
The concentration of
HFABP is 10 fold lower in skeletal muscle than cardiac muscle, and the amount
in kidney, liver and small intestines are lower again.65, 66
27
Functions
It is involved in the intracellular uptake and buffering of free fatty acids
in the myocardium.67
It has diverse role in fatty acid metabolism, trafficking
and signalling. The continuous and profound demand of cardiac muscle
contraction are associated with a wide range of specialized metabolic
adaptations which includes preferential use of fatty acids as energy substrate.68
HFABP binds long chain fatty acids, their acyl and carnitine derivatives with
1:1 stoichiometry and high affinity.69,70
It helps in the uptake of fatty acids and
energy production.
Clinical Significance
HFABP is abundant in myocardium and rapidly released from
cardiomyocytes into blood. It appears in plasma within 2 hours of cardiac
damage, peaks within 4 to 6 hours and returns to normal basal level by 20
hours.71
Because of its low molecular weight, relative tissue specificity and
high myocardial content, this marker is released earlier than other markers.
HFABP is the earliest biomarker available for acute myocardial injury.72
It has
been proven to be an independent prognostic marker in patients with MI.73
28
AIMS AND OBJECTIVES
Aims
To estimate the serum level of Heart type Fatty Acid Binding Protein
(H-FABP) in Patients within 6 hours of onset of chest pain.
Objectives
1. To correlate H-FABP Level with CK-MB
2. Correlation of H-FABP with Lipid Profile
1. Total cholesterol
2. High density Lipoprotein(HDL)
3. Low density Lipoprotein (LDL)
4. Very Low density Lipoprotein (VLDL)
5. Triacylglycerol (TAG)
29
MATERIALS AND METHODS
After getting approval from the Institutional Ethical Committee, this
study was conducted at K.A.P.V. Govt. Medical College and MGMGH,
Tiruchirappalli during the period of June 2015 to June 2016.
This age matched cross sectional study included 90 subjects with age
limit of 30 to 60 years. Out of 90, 45 were study group, 45 were control group.
Study group were selected from patients admitted with in ICCU with
complaints of chest pain and diagnosed to have coronary artery disease and 45
healthy individuals as controls. Informed and written consent were obtained
from both study and control group.
Inclusion Criteria
1. Chest pain of duration less than 6 hours
2. ECG showing abnormal ST-T Segment changes- ST elevation or
depression, T wave inversion
Exclusion Criteria
1. Hepatic disease
2. Renal disorders
3. Heart failure
4. Pulmonary oedema
5. Cardiomyopathy
6. Stroke
7. Patient underwent CABG, Coronary Angioplasty within 30 days.
30
Sample Collection
Under aseptic precautions, 4 ml of blood was collected in a tube
containing procoagulant from study and control group by venepuncture. Blood
samples were centrifuged at 1000 X g for 15 minutes. Serum were separated
from the cells and divided into two portions.
One portion of serum was used for measuring serum Urea, Creatinine,
Uric acid, Aspartate Transaminase and CK-MB. Another portion of serum was
stored at -19°C for estimation of H-FABP.
Fasting blood samples were collected from patients and controls for the
estimation of serum Glucose, Total Cholesterol, Triglycerides and HDL.
Analysis
The following parameters were estimated by using ELISA reader, Fully
and Semi Automated Analysers.
31
S.NO ANALYTE METHOD
1
Heart type of Fatty Acid Binding Protein
(H-FABP)
ELISA
2 CK-MB
Immuno Inhibition
Method
3 FBG
Trinders, Endpoint, Fixed
time
4 Urea Urease GLDH Method
5 Creatinine Jaffe’s Kinetic Method
6 Aspartate Transaminase UV Kinetic Method
7 Uric acid Uricase Method
8 Total Cholesterol CHOD-PAP Method
9 Triglyceride GPO-PAP Method
10 HDL-C
Selective inhibition
Method
(Direct Method)
The following parameters were calculated by using FRIEDEWALDS formula.
VLDL = TGL / 5
LDL = TC – (HDL + TGL / 5)
32
1. Human serum Heart type Fatty Acid Binding Protein (HFABP)
Estimation:
Principle:
HFABP was measured by Quantitative Sandwich Elisa Method.
Antibody specific for HFABP coated wells were supplied. Sample and
standards were added into the wells. The antigen present in the samples bound
with the antibody and formed antigen antibody complex. Second antibody-
Biotinylated antibody was added into the wells. This biotinylated antibody
bound with antigen antibody complex, produces sandwich. Unbound
biotinylated antibodies were washed away. HRP-Conjugated streptavidin was
added. This enzyme bound with the sandwich complex. TMB Substrate was
added. The enzyme bound with antigen antibodies complexes reacted with the
substrate, produces colour. Stop solution was added finally. The intensity of
colour is directly proportional to the concentration of antigen present in the
sample.
Reagents:
1. FABP-3 Microplate ------------ 96 wells (12 strips x 8 wells) coated
with anti- Human FABP-3.
2. Wash Buffer Concentrate ------------ 25 ml of 20X concentrated solution
3. Standard Protein ------------- 2 vials of Human HFABP
4. Detection Antibody FABP-3 ----------- biotinylated antihuman HFABP
5. HRP-Streptavidin Concentrate --------- 200 µl 600X concentrated HRP-
conjugated Streptavidin
33
6. TMB One-Step Substrate Reagent -------- 3, 3, 5, 5'-tetramethylbenzidine
(TMB) in buffer solution.
7. Stop Solution -------- 8 ml of 0.2 M sulfuric acid.
8. Assay Diluent D -------- 15 ml of 5x concentrated buffer.
9. Assay Diluent B -------- 15 ml of 5x concentrated buffer.
Storage
The entire kit was stored at -20°C.
Reconstituted reagents – detection antibody concentrate, HRP-
Streptavidin concentrate, Assay Diluent D and Assay Diluent B were stored at
4°c.
Sample collection and storage
Serum separator tube was used to collect samples. Centrifugation done
and serum was separated and stored at <-20°C. Repeated freeze thawing was
avoided.
Reagent preparation
All the reagents were brought to the room temperature before being
used. Wash buffer was brought to room temperature and waited till the crystals
had dissolved. Brief spin was applied to standard protein vial, HRP-
Streptavidin concentration and detection antibody vial before used.
34
Preparation of Assay Diluent –D
15 ml of 5x concentrated buffer was given. 1X buffer was needed. By
using the formula C1V1=C2V2 , 5 mL of 5X Concentrated buffer was added
with 20mL of Distilled water to make 1X buffer of 25mL.
Preparation of wash buffer
25 ml of 20X concentrated solution was given. 25mL of 20X
concentrated wash buffer was added with 475mL of distilled water to make 500
mL of 1X wash buffer.
Preparation of HFABP standards
Brief spin was applied to standard protein vial. 400µl of 1X Assay
Diluent –D was added into standard protein vial and mixed thoroughly to
prepare 200 ng/ml stock solution(standard 7).6 Eppendorf tubes were taken
and numbered from 1 to 6. 300µl of 1X Assay Diluent was added to each
Eppendorf tubes. 100µl was taken from stock solution and added to tube no 6
to make 400µl of concentration 80ng/ml. 200µl was taken from tube no 6 and
added to tube no 5 to make a concentration of 32ng/ml. 200µl from tube no 5
was transferred to tube no 4(12.8ng/ml). Then 200µl from tube 4 to tube 3,
from tube 3 to tube 2 with concentration of 5.12ng/ml and 2.05 ng/ml
respectively. The tube 1 contained 300 µl of Assay Diluent-D only, which
served as a blank (0ng/ml).
35
Standards Assay Diluent-D Final
concentration
Standard protein
vial
(standard 7 )
Powder 400 µl 200 ng/ml
S6 200 µl of S7 300 µl 80 ng/ml
S5 200 µl of S6 300 µl 32ng/ml
S4 200 µl of S5 300 µl 12.8 ng/ml
S3 200 µl of S4 300 µl 5.12 ng/ml
S2 200 µl of S3 300 µl 2.05 ng/ml
S1 -------- 300 µl 0 ng/ml
Preparation of Assay Diluent- B
15ml of 5X concentrated buffer was given. 5 ml of 5X concentrated
buffer was added with 20 ml of Distilled water to make 25 ml of 1X Assay
Diluent-B
Preparation of Detection Antibody Concentrate
Antibody concentrate was prepared by adding 100 µl of Assay Diluent-
B to detection Antibody vial. Antibody concentrate was 80 fold diluted. 100µl
of Antibody concentrate was added with 7900µl of Assay Diluent – B.
Preparation of HRP-Streptavidin concentrate vial
200µl of 600X concentrated HRP-Streptavidin was supplied. 20µl of
HRP-Streptavidin was added with 11.880µl of Assay Diluent-B to make a final
concentration of 1X of 12 ml.
36
Assay procedure
1. All reagents and samples were brought to room temperature.
2. Excess micro plate strips were removed from the plate frame, returned
to the plastic pouch with the desiccant pack.
3. 100 µl of each standard and samples were added into the wells.
4. Covered and incubated at room temperature for 2.5 hours with gentle
shaking.
5. Each well was Aspirated and washed with 1X wash buffer, for a total of
four washes. Liquid in the wells were completely removed after each
wash for good performance. After the last wash, the remaining buffer
was aspirated. Then the plate was inverted and blotted against clean
paper towels.
6. 100 µl of 1X prepared biotinylated antibody was added and incubated
for 1 hour at room temperature with gentle shaking.
7. Wash step was repeated.
8. 100 µl of prepared HRP conjugated Streptavidin solution was added and
incubated for 45 minutes at room temperature with gentle shaking.
9. Wash step was repeated as in step 5.
10. 100 µl of TMB substrate reagent was added, covered and incubated for
30 minutes at room temperature with gentle shaking.
11. 50 µl of stop solution was added. The optical Density was measured at
450nm immediately.
37
Data Analysis
Mean absorbance for standards and samples were obtained and plotted
on log-log graph paper with standard concentration on the X-axis and
absorbance on the Y axis.
Normal value of HFABP
The normal reference range is 0 to 6 ng/ml.
Cut-off value for AMI is >19 ng/ml.
HFABP
STANDARDS
Concentration
(ng/ml) Absorbance
S1 0 0.438
S2 2.05 0.595
S3 5.12 0.643
S4 12.8 0.675
S5 32 0.789
S6 80 0.976
S7 200 1.238
38
Graphical representation of HFABP Standard Concentration against its
Absorbance as in LOG LOG chart
39
Estimation of Serum Creatine Kinase – MB:
Methodology: Kinetic Immune Inhibition Method
Principle:
This procedure involves measurement of CK activity in the presence of
antibody to CK – M monomer. This antibody completely inhibits the activity of
CK – MM and half of the activity of CK – MB while not affecting B subunit
activity of CK – MB and CK – BB .Then the CK method is used to
quantitatively determine CK – B activity. The CK – MB activity is obtained by
multiplying the CK – B activity by two.
Creatine kinase
Creatine phosphate + ADP Creatine + ATP
Hexokinase
ATP + D – glucose Glucose -6-Phosphate + ADP
G-6-PD
Glucose -6-Phosphate + NADP + H+ Glucose-6-Phosphate+ NADPH
G-6-PD- Glucose -6-Phosphate dehydrogenase
Sample : non- haemolysed samples.
Reagents :
Reagent 1 – Buffer / enzymes and Anti human polyclonal CKM
antibody (sheep)
Reagent 2 – creatine phosphate, ADP, G-6-PD
Reagent Reconstitution – Reagents were allowed to attain the room
temperature. 4 volume of Reagent 1 was added to 1 volume of Reagent 2.
Direct exposure to light was avoided.
40
Procedure
Test
Reconstituted Working Reagent 1000 µl
Sample 40µl
Reagents were mixed well and incubated at 37 c for 100 seconds.
The absorbance change per minute was read during 5 minutes.
Absorbance was read at 340 nm.
Calculation
CK-MB activity (U/L) = OD/min X 8254
Reference values – Serum – 0 – 24 U / L
Linearity- 600U/L
Estimation of Glucose
Method : Glucose oxidase – peroxidase (GOD-POD) method.
Analysis : End Point Analysis
Principle : Glucose is oxidized to yield gluconic acid and hydrogen
peroxide in the presence of glucose oxidase. Hydrogen
peroxide oxidatively couples with 4-amino antipyrine and
phenol to produce red quinoneimine dye in the presence of
peroxidase. This red dye has maximum absorbance at
505nm. The intensity of the coloured complex is directly
proportional to the concentration of glucose in specimen.
41
D-Glucose + O2 + H2O Gluconic acid + H2O2
H2O2 + 4- amino antipyrine + Phenol red dye + H2O
Specimen: Fresh non haemolysed serum
Assay Procedure
Enzyme reagent and standard were brought to the room temperature
before performing the assay.
Reagents Blank Standard Sample
Glucose enzyme reagent 1000µl 1000µl 1000µl
Standard - 10µl -
Sample - - 10µl
Distilled water 10µl - -
The tubes were mixed thoroughly and incubated at 37°C for 10 min. The
absorbance was read against reagent blank at 505nm.
Calculation
Absorbance of test
Glucose (mg/dl) = X Concentration of standard (mg/dl)
Absorbance of standard
Glucose Standard : 100mg/dl
Linearity : Up to 500mg/dl
Normal Values : 90-120mg/dl
Glucose fasting : 60-100mg/dl
Glucose postprandial : 90-140mg/dl
42
Estimation of Blood Urea
Method: Urease – GLDH Method
Principle:
Urea in the sample is hydrolysed by urease to ammonia and carbon
dioxide. The second reaction catalysed by glutamate dehydrogenase (GLDH )
converts Ammonia and α – ketoglutarate to glutamate and water with the
concurrent oxidation of reduced NADH to NAD . Two moles of NADH are
oxidized for each mole of Urea present.
urease
Urea + H2O 2NH4+ + CO2
GLDH
NH4+ + NADH + H
+ + 2 – oxoglutarate glutamate + NAD
+
The initial rate of decrease in absorbance at 340nm is proportional to the
Urea concentration in the sample.
Reagent composition:
Reagent1: α – ketoglutaric acid 99.8 mmol/l , Urease 23.5 KU / l , GLDH 3.5
KU / l, Adenosine diphosphate 7.6 mmol/l, Sodium azide 0.2 %
Reagent2: NADH 2.95mmol / l, Sodium azide 0.1 %
Reagent Preparation:
Working Reagent was prepared by mixing 4 parts of reagent 1with one
part of Reagent 2.
43
Procedure:
BLANK STANDARD TEST
Working Reagent 1000µl 1000µl 1000µl
Distilled water
10µl - -
Standard - 10µl -
Test - - 10µl
Mixed well and the absorbance was read after 30 seconds (A1) and 60
seconds (A2) at 340nm.
Calculation:
∆A = A2 – A 1
∆A of Test
Urea (mg/dl) = X Concentration of standard (50mg/dl)
∆A of Standard
Linearity:
The method is linear up to 200mg/dl.
Reference values: 15 – 30 mg/dl.
Estimation of Serum Creatinine
Methodology : Modified Jaffe’s reaction
Principle:
Creatinine present in the sample reacts with picric acid in alkaline
medium forming Creatinine picrate (red coloured complex) which is measured
photo metrically at 505 nm.
44
Reagent composition:
Reagent 1 – picric acid reagent – 25.8mmol/L
Reagent 2 – sodium hydroxide – 95mmol/L
Creatinine standard – 2 mg / dl
Reagent Preparation:
Equal volume of Reagent 1 and 2 were mixed and allowed to wait for 15 min
before use.
Sample: non- haemolysed serum.
Assay procedure:
Tubes Working reagent Standard Test sample Distilled water
Blank 1000µl - - 100µl
Standard 1000µl 100µl - -
Test 1000µl - 100µl -
Mixed well and initial absorbance (A1) was read at 20 sec after mixing
and final absorbance (A2) after 80 sec after mixing.
Calculation:
A = A2 – A1
∆A of Test
creatinine (mg/dl) X Concentration of standard (2mg/dl)
∆A of Standard
Linearity – up to 18 mg/dl.
45
Reference Range for serum creatinine:
Males : 0.7-1.4 mg/dl
Females : 0.6-1.2mg/dl.
Estimation of Uric Acid:
Method: Uricase method
Principle:
Uric acid is acted on by uricase forming allantoin and hydrogen
peroxide. The hydrogen peroxide reacts with phenolic chromogens in presence
of peroxidase forming a red coloured compound.
Peroxidase
H2O2 + Phenolic chromogen Red coloured compound
This compound has maximum absorption at 520 (500 – 530) nm.
Absorption is directly proportional to the concentration of uric acid.
Procedure:
Incubate for 10 minutes at room temperature. After completion of
incubation, measure absorbance of acyl mixture against blank at 520nm.
Concentration of Uric acid Standard – 6mg/dl.
Reagent Sample Standard Blank
Working reagent 1ml 1ml 1ml
Sample 25 µl -- -- ---
Standard --- 25 µl ---
46
Calculation:
Amount of uric acid present in 100 ml of plasma or serum
OD test – OD blank Conc. Of std
= X X 100
OD std - OD blank Volume of sample
OD test – OD blank 0.0015
= X X 100 mg/dl
OD std - OD blank 0.025
OD (T) – OD (B)
= X 6 mg/dl
OD (S) - OD (B)
Result: The concentration of uric acid in given sample of serum = -------- mg%
Reference Range of Uric acid:
Males : 3.5 to 7 mgs %
Females : 2.5 to 6 mgs %
Estimation of S.Total Cholesterol
Method: Cholesterol oxidase-Peroxidase Enzymatic, endpoint method.
Principle: The free Cholesterol, liberated from the cholesterol esters by
cholesterol esterase, is oxidized by cholesterol oxidase to cholestenone with the
simultaneous production of hydrogen peroxide. The hydrogen peroxide reacts
with 4 amino antipyrine and a phenolic compound in the presence of
peroxidase to yield a red coloured complex.
47
ChE
1. Cholesterol ester + water cholesterol + fatty acid
ChO
2. Cholesterol + oxygen cholest-4-en-3one +H 2O 2
POD
3. 2H2O2 + 4AAP+ phenol quinoneimine dye + 4H2O
• ChE- Cholesterol esterase
• ChO- Cholesterol oxidase
• 4AAP- 4 amino antipyrine
• POD- Peroxidase
Absorbance of quinoneimine formed is directly proportional to
cholesterol concentration.
Reagent:
Reagent-1(Enzyme/chromogen)
Reagent-1A (BUFFER)
Cholesterol standard-200mg/dl
Reconstituted reagent:
Contents of one bottle of the reagent-1 is dissolved in one bottle of
buffer (reagent 1A).
48
Assay Procedure
Blank Standard Test
Working reagent 1000µl 1000µl 1000µl
Distilled water 10µl - -
Standard - 10µl -
Sample - - 10µl
Mixed well and incubated for 10 min at room temperature. The
absorbance of the test and standard were read against reagent blank at
wavelength of 505 nm.
Calculation
Absorbance of test
Cholesterol (mg/dl) = X Concentration of standard (mg/dl)
Absorbance of standard
Reference Range: 150-200 mg/dl
Linearity –up to 750 mg/dl
Sensitivity-1mg/dl
Interference:
Hb up to 200mg/dl, Ascorbate up to 12mg/dl,Bilirubin up to 10mg/dl
and Triglycerides upto700 mg/dl do not interfere with the test.
49
Estimation of S.Triglycerides
Method: GPO-PAP method, endpoint
Methodology:
Colorimetric, enzymatic method with glycerol phosphate oxidase.
This reagent is based on the method of wako and the modifications by
McGowan et al..and Fossati et al..
Principle:
LPL
Triglycerides + H2O Glycerol + free fatty acids
GK
Glycerol + ATP Glycerol 3 phosphate + ADP
GPO
Glycerol 3 phosphate + O2 DAP + H2O2
H2O2 + 4AAP + 3,5-DHBS Quinoneimine dye + 2H2O
LPL- Lipoprotein lipase
GK- Glycerol kinase
GPO- Glycerol Phosphate Oxidase
DAP-Dihydroxy Acetone Phosphate
ATP- Adenosine Tri Phosphate
4AAP- 4Amino Anti Pyrine
DHBS-3,5Dichloro-2Hydroxy Benzene Sulfonate
50
Lipoprotein lipase catalysed hydrolysis of triacylglycerol and produced
glycerol which was phosphorylated by glycerol kinase using ATP to
glycerol-3-phosphate which upon oxidation yielded Dihydroxy acetone
phosphate and hydrogen peroxide. The hydrogen peroxide reacted with
phenolic compound and 4amino antipyrine to form a coloured complex.The
intensity of Quinoneimine dye formed was proportional to the triglyceride
concentration in the sample when measured at 505 nm (500-540nm).
Reagent:
Reagent 1(Enzymes/chromogen)
Reagent 2(Buffer)
Triglycerides standard concentration- 200mg/dl
Reagent preparation:
The working reagent was prepared by mixing 4 parts of R1 with 1 part of
R2.Stable for 90 days at 2-8 ◦C.
Sample: Non haemolysed serum collected after 12 hrs of fasting.
Assay procedure
Pipette into tubes
marked
Blank
Standard
Test
Working reagent
1000µl
1000µl
1000µl
Distilled water
10µl
-
-
Standard
-
10µl
Sample
-
-
10µl
51
Mixed and incubated for 10min at room temperature .Absorbance were
read at 505nm/670nm for standard and sample against reagent blank.
Calculation:
Absorbance of test
Triglycerides (mg/dl) = X Concentration of standard (mg/dl)
Absorbance of standard
Reference values
Serum /plasma fasting level: 25-160mg/dl
Linearity – up to 1000mg/dl
Sensitivity- 2mg/dl
Interference:
Hb up to300mg/dl,
Ascorbate up to 3mg/dl,
Bilirubin up to 20mg/dl
Estimation of S.HDL Cholesterol
Method: Selective Inhibition Method (Direct Method).
Principle:
The reaction between cholesterol other than HDL and the enzyme for
Cholesterol assay is suppressed by the electrostatic interaction between
polyanions& cationic substances. Hydrogen peroxide is formed by the free
cholesterol in HDL by cholesterol oxidase. Oxidative condensation of EMSE
and 4-AA is caused by Hydrogen peroxide in the presence of peroxidase, and
52
the absorbance of the resulting red-purple quinone is measured to obtain the
cholesterol value in HDL.
Polyanions
Other lipoprotein than HDL suppress reaction with enzyme
Cationic substances
Cholesterol esterase
HDL (Cholesterol esters) + H2O HDL (free cholesterol)+ FFA
Cholesterol oxidase
HDL (Cholesterol esters) + O2 + H+
cholestenone + H2O2
Peroxidase
2H2O2 + 4-AA + EMSE + H3 + O violet quinone + 5H2O
Reagent Composition
Reagent 1: HDL-C DIRECT – N-ethyl- N-(3-methyl phenyl)- N
Succinylethynediame(EMSE)
Reagent 2: cholesterol oxidase
4-Amino antipyrine
HDL cholesterol standard – 25mg/dl
Sample: Non haemolysed serum
53
Procedure:
Blank Calibrator Sample
Reagent 1 450µl 450µl 450µl
Distilled water 5 µl
Calibrator 5 µl
Sample 5 µl
Mixed Well And Incubated For 5 Minutes At 37°C
Reagent 2 150 µl 150 µl 150 µl
Mixed Well And Incubated For 5 Minutes At 37°C
Absorbance was read at 630 nm.
Calculation
Absorbance of test
HDL cholesterol (mg/dl) = x concentration of calibrator
Absorbance of calibrator
Absorbance of the test
= x 25
Absorbance of the calibrator
Linearity-up to 150mg/dL
Normal Values:
Males - 35 to 80mg/dl
Females - 42 to 88mg/dl
Freidwald’s formula for calculation of LDL
VLDL=TGL/5,if TGL is less than 400mg/dl
LDL = Total cholesterol – (HDL + VLDL)
54
Estimation Aspartate Aminotransferase of Serum
Methodology: Modified IFCC Method
Principle
The transfer of amino group from L-Aspartate to α- ketoglutarate to
yield oxaloacetate and L – Glutamate is catalysed by AST. When Oxaloacetate
undergoes reduction there is simultaneous oxidation of NADH to NAD in the
presence of MDH.
The decrease in the rate of absorbance at 340nm is directly proportional
to the AST activity. Interference from endogenous pyruvate which is normally
present in serum is prevented by the addition of LDH.
AST
L-Aspartate + α- ketoglutarate Oxaloacetate +L-glutamate
MDH
Oxaloacetate + NADH +H Malate + NAD
Reagent Composition:
R1:
Tris Buffer (pH 7.8) - 20mmol/L
L-Aspartate - 230mmol/L
LDH - > 33.3µkat/L
2- Oxoglutarate - 13.21mmol/L
MDH - 3.333 µkat/L
Also contains Non –reactive fillers and stabilizers.
R2:
NADH - 1.51mmol/L
55
Reagent Preparation
Prepare the working reagent by mixing 4 parts of R1 with 1 part of R2
per Assay tube.
Assay Procedure
Volumes
Working Reagent 1000µl
Test 100 µl
Mixed well and incubated for 1 minute at 37 c. change in absorbance
per minute was read during 3 minutes.
Calculation
AST ACTIVITY (U/L) = OD/min X 1745
Limitations
1. Sample with values above 1600 IU/L should be diluted 1:1 eithsaline, re
assayed and the results multiplied by two.
2. Patients with severe vitamin B6 deficiency could have a decreased
recovery of AST, presumably due to a lack of pyridoxal phosphate.
Linearity
AST reagent is linear up to 1600 U/L. For values above the linearity
limit dilute with saline and re assay. Multiply by the dilution factor to obtain
the end result.
Expected values
Adult Male :< 35 U/L
Adult Female :< 31 U/L.
56
RESULTS
A total of 90 subjects were selected for the present study. This includes
45 cases with MI and 45 healthy controls. Statistical analysis was done using
SPSS-16. Student t test was employed for statistical analysis of data. Mann
Whitney test was also performed because of wide standard deviation.
Correlation between the measured parameters was done by using Pearson’s
correlation. The data were expressed in terms of mean and standard deviation.
p value <0.05 was taken as significant. The biochemical values obtained for
cases and controls are presented in master chart I and II respectively.
57
MASTER CHART FOR CASES-I
SNO AGE DM HT SMOKE ALCO
HOL SBP DBP GLU UREA
CREA
TININE CHO TGL HDL LDL VLDL AST
URIC
ACID CKMB HFABP
DURA
TION
1 55 N N N N 100 70 230 29 0.8 226 153 32 163.4 30.6 47 6.2 31 32.6 5.3
2 35 N N Y Y 150 90 179 31 1.4 230 148 36 164.4 29.6 87 8.9 44 67.9 4
3 38 N N Y Y 140 90 97 17 0.8 130 102 40 69.6 20.4 98 8.5 18 40.3 4.3
4 50 N N Y Y 130 80 139 30 1.1 224 184 42 145.2 36.8 29 7.1 5 28.3 4
5 43 N N N N 110 70 175 20 0.9 198 242 38 111.6 48.4 19 4.2 40 34.4 3
6 40 N N Y Y 90 60 116 43 1.6 170 160 45 93 32 39 6.1 24 28.3 5
7 45 N N Y N 120 80 188 21 0.7 253 227 40 167.6 45.4 34 10.6 50 27.9 2
8 60 Y N N N 140 80 85 24 0.6 210 145 19 162 29 57 8.5 42 49.5 4
9 55 N N Y Y 140 90 86 43 1.3 270 152 39 200.6 30.4 295 7.7 146 34.3 6
10 60 N N Y Y 130 90 87 34 1.9 270 127 40 204.6 25.4 69 8.6 86 46.7 4
11 36 N N Y Y 180 80 185 33 1.2 194 157 45 117.6 31.4 132 7.9 112 49.3 4
12 44 N Y N N 130 70 272 56 3 261 143 40 192.4 28.6 133 5.2 15 17.1 3
13 41 Y Y N N 100 70 142 42 1.1 249 112 35 191.6 22.4 113 4.4 96 20.7 5
14 60 N N Y Y 120 60 132 38 1 166 159 32 102.2 31.8 25 6.2 55 26.4 4
15 42 N N Y Y 160 90 53 21 1.1 149 126 38 85.8 25.2 33 4 78 32.6 4
16 40 N N Y N 150 90 65 36 1.5 162 142 40 93.6 28.4 20 7.5 48 51.8 3
17 52 N N Y Y 130 80 283 18 0.7 176 135 39 110 27 132 8.8 8 22.1 1
18 38 N N N N 140 90 100 19 0.7 156 114 35 98.2 22.8 90 7.2 40 29.6 4
19 53 N N N N 170 100 91 38 1.6 234 128 74 134.4 25.6 86 4 61 18.2 5
20 46 N N Y Y 120 90 83 26 0.8 179 108 40 117.4 21.6 37 4.2 36 33.4 3
21 38 Y Y N Y 130 90 147 34 1.5 264 136 42 194.8 27.2 34 4.2 36 31.6 3.3
22 46 Y Y N N 128 86 312 39 1.1 186 137 40 118.6 27.4 72 3 30 8.2 2
23 50 N N Y N 150 100 131 28 1.4 215 222 43 127.6 44.4 55 8.4 67 26.6 3
58
MASTER CHART FOR CASES-I
24 60 Y N Y Y 90 60 81 42 1.7 171 142 39 103.6 28.4 32 9.2 45 33 2
25 33 N N Y N 100 70 180 18 4.7 224 176 32 156.8 35.2 374 9.3 86 41.8 5
26 54 Y N N Y 160 80 133 26 1.4 174 149 42 102.2 29.8 64 8.5 48 37.4 3.3
27 44 Y Y Y Y 130 70 280 39 1.1 227 187 31 158.6 37.4 43 10.5 76 30.1 4
28 56 N N Y N 90 60 325 20 1.2 162 187 30 94.6 37.4 66 8.8 10 41.6 3
29 41 N Y Y N 100 80 326 46 1.4 237 178 37 164.4 35.6 89 11.8 80 22 3
30 33 Y Y N N 110 90 152 79 2.5 187 132 42 118.6 26.4 38 7.9 50 11.5 6
31 45 N N Y Y 80 60 229 41 1.2 198 174 40 123.2 34.8 41 7.7 16 59.9 5
32 45 Y N Y N 140 70 104 20 0.7 221 165 38 150 33 52 8.9 230 65.1 5
33 55 N N Y N 130 70 80 32 0.8 206 111 38 145.8 22.2 30 6.2 17 51.8 5
34 38 Y N N N 100 60 88 26 1 198 145 32 137 29 192 8.9 18 32.5 2
35 55 Y N Y Y 110 80 86 26 1.4 189 143 45 115.4 28.6 96 6.5 38 8.7 4
36 50 N N Y Y 120 70 131 28 1.4 215 122 43 147.6 24.4 43 4.5 9 27.2 2
37 32 Y N N N 120 80 104 35 1.2 253 156 35 186.8 31.2 181 9.3 138 49.4 4
38 40 N N Y N 130 90 132 35 0.9 156 134 45 84.2 26.8 26 7.7 14 13.5 2
39 47 Y N N N 140 90 118 58 1.8 224 166 34 156.8 33.2 67 6.5 98 44.1 4
40 35 Y N Y N 120 70 110 67 2 246 156 32 182.8 31.2 128 12 23 5.6 3
41 59 N N N N 130 80 267 38 1.3 215 164 27 155.2 32.8 263 5.7 66 43.2 6
42 42 Y N Y N 90 60 394 86 1.1 277 149 46 201.2 29.8 36 4.9 38 62.3 3
43 47 N N Y Y 130 100 186 26 0.9 324 158 42 250.4 31.6 36 8.2 45 32 4
44 40 N N Y N 140 90 78 36 1 165 145 34 102 29 28 5.9 78 115.5 6
45 35 N N N Y 160 94 108 45 1.1 276 175 32 209 35 54 8.7 22 25.5 3
59
MASTER CHART FOR CONTROLS –II
SNO AGE DM HT SMOKE ALCO
HOL SBP DBP GLU UREA
CREA
TININE
CHO TGL HDL LDL VLDL AST URIC
ACID CKMB HFABP
1 56 N N Y Y 130 90 68 18 1.1 186 99 36 130.2 19.8 23 3.5 6 4.7
2 48 Y Y Y Y 140 90 94 22 1 145 135 40 78 27 13 7.3 14 2.2
3 48 N N N N 120 80 84 17 1 161 147 42 89.6 29.4 16 3.9 15 3.9
4 42 N N N N 120 80 96 21 1 141 92 51 71.6 18.4 12 4.1 11 5.2
5 38 Y N Y N 110 70 86 25 0.9 207 154 35 141.2 30.8 17 4.8 15 2.3
6 41 N N Y N 128 86 92 13 0.8 179 126 41 112.8 25.2 23 4.4 13 1.4
7 51 y N Y N 120 80 86 13 0.8 177 142 40 108.6 28.4 17 4.3 18 1.9
8 52 y N Y Y 110 70 94 17 0.8 129 126 35 68.8 25.2 19 5.4 20 0.7
9 52 N y N N 110 70 85 22 1 152 119 48 80.2 23.8 26 5.6 14 5.3
10 31 N N N N 100 70 72 16 0.8 137 149 47 60.2 29.8 20 5.3 9 3.4
11 34 y N N N 110 70 90 19 0.8 131 111 31 77.8 22.2 20 5.3 14 2.8
12 52 N y Y Y 140 70 89 21 1.1 184 123 47 112.4 24.6 19 5.6 17 4.9
13 54 Y N N N 120 70 97 15 0.9 169 150 32 107 30 16 5.7 10 3.6
14 46 y y N N 130 80 120 14 0.9 197 115 40 134 23 18 5.9 12 3.7
15 38 N N Y Y 110 80 107 20 0.8 177 139 44 105.2 27.8 15 5.6 16 0.9
16 42 Y N Y N 120 80 78 23 0.9 164 135 43 94 27 18 4.9 11 4.9
17 60 N Y Y Y 130 90 73 11 0.7 174 147 41 103.6 29.4 10 5.2 17 0.9
18 40 N N Y N 140 90 67 10 0.7 173 126 56 91.8 25.2 21 5 8 5.5
19 37 N N N Y 100 70 83 22 0.9 149 73 38 96.4 14.6 12 4.6 10 4
20 36 N N N Y 110 80 90 30 1.2 184 117 49 111.6 23.4 11 6.7 15 2
21 33 N N N N 120 80 80 21 1.1 154 92 37 98.6 18.4 15 4.4 15 2.3
22 42 N N N N 120 80 75 21 1.1 202 122 32 145.6 24.4 27 5.7 12 4.1
23 35 N N Y N 130 80 69 17 1.7 182 81 47 118.8 16.2 12 5.3 10 3.5
60
MASTER CHART FOR CONTROLS –II
24 43 N N Y N 120 90 77 25 1.2 174 129 34 114.2 25.8 20 5.3 8 1.3
25 43 N N N N 100 70 76 23 0.8 196 69 55 127.2 13.8 13 4.8 16 0.5
26 56 Y N Y N 110 70 99 21 1 152 143 31 92.4 28.6 27 5 12 3.4
27 52 Y Y N N 130 70 86 19 1 149 80 38 95 16 7 2 13 1.7
28 36 N N Y N 120 80 96 13 0.8 152 89 41 93.2 17.8 20 2.8 15 3.5
29 45 N N N N 120 80 91 13 0.8 145 101 51 73.8 20.2 13 3.5 23 1.1
30 47 Y N N N 120 80 71 12 0.6 160 104 42 97.2 20.8 23 2.9 14 2.5
31 54 Y Y N N 110 70 86 14 0.7 180 128 54 100.4 25.6 15 3 10 1.8
32 36 Y N Y Y 130 70 91 16 0.8 175 92 49 107.6 18.4 22 3.4 8 3
33 47 N N Y N 120 80 70 18 0.6 156 99 37 99.2 19.8 23 3.1 17 2.9
34 50 N Y Y Y 120 80 106 19 0.7 167 147 44 93.6 29.4 14 5 14 3.2
35 54 N N Y N 120 80 99 16 0.7 201 142 50 122.6 28.4 20 3.4 8 1.4
36 42 N N N N 100 70 127 16 0.8 193 128 41 126.4 25.6 18 3.7 12 3
37 39 Y Y N N 128 80 81 13 1.1 178 83 35 126.4 16.6 29 5.3 10 6.1
38 33 N N Y N 120 80 80 16 1.2 186 130 45 115 26 16 4.8 13 2.1
39 46 N N N N 130 80 98 23 1.1 171 83 37 117.4 16.6 20 5.3 18 2
40 49 N N Y Y 120 80 139 28 1.2 198 134 29 142.2 26.8 9 7.4 16 2.2
41 38 N N Y N 100 70 86 17 1.1 184 144 39 116.2 28.8 21 4.9 7 0.8
42 58 N Y Y N 110 80 88 19 1.2 165 150 45 90 30 23 4.8 20 1.6
43 45 Y N N N 130 90 79 12 0.9 139 85 40 82 17 18 3.2 16 4.2
44 32 N N Y N 120 80 80 18 0.9 189 120 43 122 24 19 4.8 14 1.4
45 53 Y Y N N 90 60 79 19 0.8 193 71 59 119.8 14.2 14 1.8 22 1.6
61
Table -1
Descriptive Statistics of Control and Study Group
Variance
Control Group
(n=45)
Study Group
(n=45)
Min. Max. Mean S.D Min. Max. Mean S.D
AGE 31.0 60.0 44.57 7.81 32.0 60.0 45.62 8.35
SBP 90.0 140.0 118.58 11.51 80.0 180.0 126.17 22.98
DBP 60.0 90.0 77.68 7.22 60.0 100.0 79.33 12.16
GLUCOSE 67.0 139.0 88.00 14.92 53.0 394.0 157.11 83.10
UREA 10.0 30.0 18.17 4.50 17.0 86.0 35.31 15.08
CREATININE .6 1.7 .93 .20 .6 4.7 1.32 .69
CHOLESTEROL 129.0 207.0 170.15 20.51 130.0 324.0 211.48 41.91
TGL 69.0 154.0 117.13 25.38 45.0 242.0 150.51 33.82
HDL 29.0 59.0 42.02 7.15 19.0 74.0 38.44 7.66
LDL 60.2 145.6 104.70 20.45 69.6 250.4 142.94 41.48
VLDL 13.8 30.8 23.42 5.07 9.0 48.4 30.10 6.76
AST 7.0 29.0 17.86 5.00 19.0 374.0 82.55 75.19
URIC ACID 1.8 7.4 4.63 1.22 3.0 12.0 7.31 2.15
CK MB 6.0 23.0 13.51 3.94 5.0 230.0 53.71 42.93
HFABP .5 6.1 2.78 1.45 5.6 115.5 35.81 19.28
62
Table - 2
Age Distribution in Control and Study Group
Age
Control Study Total
(n = 45) (100%) (n = 45) (100%) (n = 90)
(100%)
30 to 40 years
15 33.3% 15 33.3% 30 33.3%
41 to 50 years
17 37.8% 17 37.8% 34 37.8%
51 to 60 years
13 28.9% 13 28.9% 36 28.9%
Fig: 1- Age Distribution in Control and Study Group
STUDY
CONTROL
0
5
10
15
20
31 to 40 41 to 50 51 to 60
1517
13
1517
13
AGE DISTRIBUTION
STUDY CONTROL
63
Table- 3
Comparison of Age in Study and Control Group
Age Mean S.D
Statistical
Inference
Control
(n = 45)
44.578
7.8117 t = 0.613
df =88
p value 0.542
Study (n = 45)
45.578
8.3511
Comment
The Mean age of study and control group were 45.578 ± 8.3511 and 44.578 ±
7.811 respectively. The p value between the study and control group was 0.542. This
shows that there was no statistical significance of age between control and study
group. Hence these groups are comparable.
Fig: 2- Mean And S.D of Age in Study and Control Group
STUDY
CONTROL
MEAN S.D
45.578
8.3511
44.578
7.8117
STUDY CONTROL
64
Table - 4
Statistical Analysis of HFABP Level in Control and Study Group
HFABP Mean S.D
Statistical
Inference
Control (n = 45)
2.787
1.4556 t = 11.45
df =88
p value 0.000*
Study
(n = 45)
35.811
19.2871
* Significant at 0.05 level
Comment:
The Mean value of HFABP in study and control groups were 35.811 ± 19.2871
and 2.787 ± 1.4556. Mean value of study group was higher than control group and the
p value was 0.000 (<0.05) which was statistically significant. Because of the wide
standard deviation, Mann- whitney test is better than independence t test to compare
the groups.
65
Table- 5
Mann Whitney U Test for Comparison of HFABP
HFABP
Mean rank
Sum of ranks
Statistical Inference
Control
(n = 45)
67.98
3059.00
Z= -8.163
p value 0.000*
Study
(n = 45)
23.02
1036.00
* Significant at 0.05 level
Comment:
Mean rank of study and control group were 23.02 and 67.98. p value was
0.000(<0.05). This showed that the difference was statistically significant.
Fig: 3 - Mean and S.D Of HFABP in Study and Control Groups
0
5
10
15
20
25
30
35
40
MEAN S.D
35.811
19.2871
2.787 1.4556
HFABP
STUDY CONTROLS
66
Table -6
Statistical Analysis of CK-MB Value in Control and Study Group
CK-MB Mean S.D
Statistical
Inference
Control (n = 45)
13.511 3.9464
t = 6.255
df =88
p value 0.000*
Study
(n = 45)
53.711 42.9321
* Significant at 0.05 level
Comment:
Mean value of CK-MB in study group was 53.711 ± 42.9321 which was higher
than control group whose mean value was 13.511 ± 3.9464. p value of 0.000 showed
statistical significance. Mann Whitney U test was performed due to wide standard
deviation.
67
Table - 7
Mann Whitney U Test for Comparison of CK-MB
CK-MB Mean rank Sum of ranks
Statistical
Inference
CONTROL
(n = 45)
28.03
1261.50
Z = -6.348
p value 0.000*
STUDY
(n = 45)
62.97
2833.50
* Significant at 0.05 level
Comment:
The mean rank of CK-MB in study and control group were 28.03 and 62.97. p
value was 0.000 which was statistically significant.
Fig: 4 Mean and S.D of CK-MB in Study and Control Groups
0
10
20
30
40
50
60
MEAN S.D
53.711
42.9321
13.511
3.9464
CK-MB
STUDY CONTROLS
68
Table - 8
Statistical Analysis of Uric Acid Level in Control and Study Group
Uric Acid Mean S.D
Statistical
Inference
Control (n = 45)
4.638 1.2239
t = 7.228
df =88
p value 0.000*
Study
(n = 45)
7.311 2.1581
* Significant at 0.05 level
Fig: 5 Mean and S.D of Uric Acid in Study and Control Groups
Comment:
Mean value of Uric acid in study and control group were 7.311 ± 2.1581 and
4.638 ± 1.2339. p value was 0.000 and the difference between study and control group
was statistically significant.
STUDY
CONTROL
0
2
4
6
8
MEAN S.D
7.311
2.1581
4.638
1.2239
URIC ACID
STUDY CONTROL
69
Table - 9
Statistical Analysis of Lipid Parameters in Control and Study Group
Variables
Mean S.D
Statistical
Inference
Total
Cholesterol
Control
(n = 45)
170.156 20.5138
t = 5.941, df
=88
p value 0.000* Study
(n = 45)
211.489 41.9180
TGL
Control
(n = 45)
117.133 25.3822 t = 6.103, df
=88
p value 0.000* Study
(n = 45)
152.733 29.7836
HDL
Control
(n = 45)
42.022 7.1589
t = 2.288, df
=88
p value 0.025* Study
(n = 45)
38.444 7.6680
LDL
Control
(n = 45)
104.707 20.4583 t = 5.515, df
=88
p value 0.000* Study
(n = 45)
142.498 41.1642
VLDL
Control
(n = 45)
23.427 5.0764
t = 6.103, df
=88
p value 0.000* Study
(n = 45)
30.547 5.9567
* Significant at 0.05 level
70
Comment:
The above table showed that Mean value of total cholesterol, TGL. HDL, LDL
and VLDL in study group were higher than the control group. p value of Total
cholesterol, TGL,HDL, LDL and VLDL were 0.000, 0.000, 0.025. 0.000 and 0.000
respectively, showed the difference was statistically significant.
Fig: 6-Mean Value of Lipid Profile in Study and Control Groups
0
50
100
150
200
250
TC TGL HDL LDL VLDL
211.489
152.733
38.444
142.498
30.547
170.156
117.133
42.022
107.707
23.427
STUDY CONTROL
71
Table - 10
Statistical Analysis of HFABP in Control and Study Group Aged 31 To 40 Years
HFABP Mean S.D
Statistical
Inference
Control
(n = 15)
2.907 1.5126
t = 2.336, df =28
p value 0.027* Study (n = 15)
39.607 26.8245
* Significant at 0.05 level
Comment:
Mean value of HFABP in study and control groups aged 31 to 40 years were
compared. The mean value of these groups were given in the above table. Statistical
analysis showed significant difference between study and control group.
72
Table -11
Statistical Analysis of HFABP in Control and Study Group Aged 41 To 50 Years
HFABP Mean S.D
Statistical
Inference
Control (n = 17)
2.841 1.3661
t = 7.999, df =32
p value 0.000*
Study
(n = 17)
33.641 15.8178
* Significant at 0.05 level
Comment:
Mean value of HFABP in study and control groups aged 41 to 50 years were
compared. p value showed that the difference between these two groups were
statistically significant.
73
Table - 12
Statistical Analysis of HFABP in Control and Study Group Aged 51 To 60 Years
HFABP Mean S.D
Statistical
Inference
Control (n = 13)
2.577 1.5943
t = 8.864, df =24
p value 0.000*
Study
(n = 13)
34.269 12.7918
* Significant at 0.05 level
Comment:
Mean value of HFABP in study and control groups aged 51 to 60 years were
given in the above table. p value between study and control groups showed
statistically significant difference.
74
Table - 13
Statistical Analysis of HFABP Level Between Smokers and Non Smokers in
Study Group
HFABP
Variable Mean S.D Statistical
Inference
Smokers (n = 29)
38.828
21.694
t = 1.429, df =43
p value 0.160
Non-Smokers
(n = 16)
30.344
12.773
Comment:
The statistical analysis of HFABP in smokers and non-smokers were
insignificant. There were no significant difference of HFABP level between these two
groups.
Fig: 7 -Mean Value of HFABP between Smokers and Non-Smokers in Study
Group
0
10
20
30
40
SMOKERS NON SMOKERS
38.828
30.344
HFABP
75
Table - 14
Statistical Analysis of HFABP Level Between Alcoholics and Non Alcoholics in
Study Group
HFABP
Variables Mean S.D Statistical
Inference
Alcoholics (n =20)
34.760
13.192
t =-.327, df =43
p value 0.745
Non-Alcoholics
(n = 25)
36.66
23.29
Comment:
Analysis of HFABP levels between alcoholics and non-alcoholics in table 16. p
value >0.05. Hence the difference between these groups were statistically
insignificant.
Fig: 8- Mean Value of HFABP between Alcoholics and Non-Alcoholics in
Study Group
33
34
35
36
37
ALCOHOLICS NON ALCOHOLICS
34.76
36.66
HFABP
76
Table -15
Statistical Analysis of HFABP Level Between Diabetics and Non Diabetics in
Study Group
HFABP
Mean S.D Statistical
Inference
Diabetics (n = 15)
32.647 19.18
t = -.775, df =43
p value 0.433
Non-Diabaetics
(n = 30)
37.39
19.46
Comment:
Mean value of HFABP between diabetics and non-diabetics were given in
Table18. The p value of HFABP between diabetics and non-diabetics were 0.433 and
0.138 which was statistically insignificant.
Fig: 9- Mean Value of HFABP between Diabetics and Non-Diabetics in Study
Group
30
31
32
33
34
35
36
37
38
DIABETIC NON-DIABETIC
32.647
37.39
HFABP
77
Table -16
Statistical Analysis of HFABP Level between Hypertensive and Non
Hypertensive Patients in Study Group
HFABP
Variables Mean S.D
Statistical
Inference
Hypertension (n =7)
20.171 8.76
t = -2.466, df =43
p value 0.018*
No Hypertension
(n = 38)
38.692
19.36
* Significant at 0.05 level
Comment:
Table 20 shows HFABP value in hypertensives and non- hypertensives. The
mean value of HFABP in non -hypertensives was higher than hypertensives and the p
value was 0.018 which was statistically significant. This could be due to the unequal
number of subjects in these groups.
Fig: 10 - Mean Value of HFABP between Hypertensives and Non-Hypertensives
in Study Group
0
10
20
30
40
HYPERTENSION NON HYPERTENSION
20.171
38.692
HFABP
78
Table -17
Frequency table for Duration of Chest Pain and Serum Levels of HFABP, CK-
MB in Study Group
Variables
Hours
Total
< 3 hours 3 to 6 hours
count % count % count %
HFABP
(ng/mL)
Below
19 ng/mL
4 23.5% 3 10.7% 7 15.6%
Above
19 ng/mL
13 76.5% 25 89.3% 38 84.4%
CK-MB
(U/L)
Below
24 U/L
7
41.2% 4 14.3% 11 24.4%
Above
24 U/L
10 58.8% 24 85.7% 34 75.6%
Comment:
Table 22 shows frequency distribution of HFABP and CK-MB between study
and control groups. Total duration (0 to 6 hours) was divided into 2 groups- 0 to 3
hours and 3 to 6 hours. Out of 45 patients 38 patients showed elevated HFABP. Out of
this 38, 13 patients had elevated HFABP in the first 3 hours which is about 76.5% and
25 patients were in the 3 to 6 hours of duration.34 patients had elevated CK-MB,
from which 10 patients had elevated value in the first 0 to 3 hours, remaining patients
had high level in 3 to 6 hours.
When compared with CK-MB, HFABP showed 76.5% detection in first 3
hours where as CK-MB had only 58.8%. During 3 to 6 hours, the percentage of
increased HFABP and CK-MB were 89.3% and 85.7%. There was no significant
difference between HFABP and CK-MB in the 3 to 6 hours of duration.
79
Table -18
Pearson Correlation between HFABP with other Parameters in Study Group
HFABP
Variables
Correlation
value
p value Statistical
Inference
Duration
0.365 0.014* S
Smoke -0.213 0.160 NS
Alcohol
0.050 0.745 NS
DM
0.117 0.443 NS
HT
0.352 0.018* S
CK-MB
0.318 0.033* S
Total
Cholesterol
-0.056 0.714 NS
TGL
-0.030 0.845 NS
HDL
-0.217 0.152 NS
LDL
-0.012 0.939 NS
VLDL
0.020 0.896 NS
AST
0.048 0.754 NS
Uric Acid
0.015 0.920 NS
* Significant at 0.05 level
Comment:
Table 23 shows Pearson correlation between HFABP With other parameters.
There was a significant positive correlation present between HFABP with duration,
Hypertension and CK-MB.
80
Fig: 11 –Correlation between HFABP and Duration
Fig: 12 –Correlation between HFABP and CK-MB
81
Table -19
Pearson Correlation between CK-MB with other parameters in Study Group
CK-MB
Variables Correlation
Value
p value Statistical
inference
Duration
0.425 0.004** S
Smoke
-0.031 0.840 NS
Alcohol
0.132 0.388 NS
DM
-0.225 0.138 NS
HT
-0.010 0.947 NS
HFABP
0.318 0.033* S
Total
Cholesterol
0.226 0.135 NS
TGL
0.113 0.461 NS
HDL
-0.040 0.793 NS
LDL
0.218 0.151 NS
VLDL
0.107 0.484 NS
AST
0.299 0.046* S
Uric Acid
0.151 0.322 NS
* Significant at 0.05 level
** Significant at 0.01 level
Comment:
Table 24 shows Pearson correlation between CK-MB with other parameters.
Significant positive correlation was present between CK-MB with duration, AST and
HFABP.
82
Fig: 13 –Correlation between CK-MB and Duration
Fig: 14 –Correlation between CK-MB and AST
83
DISCUSSION
Ischemic Heart Disease is the most common cause of cardiovascular morbidity
and mortality. Imbalance between the myocardial oxygen supply and demand leads to
ischemia, injury and finally myocardial necrosis (infarction). Early detection of AMI
at the ischemic stage is important to prevent further progression of the disease. ECG
plays an important role in the diagnosis of AMI. But 10 to 20% of ECG shows
normal findings in CAD.74
In these cases, cardiac markers help in the diagnosis.
Cardiac markers like CK-MB, Troponins tend to elevate after 6 hours of onset of the
event. Even though Myoglobin increases within 2 hours of onset, the specificity
towards myocardium is very low. HFABP an ischemic marker, tends to elevate
earlier than other cardiac markers.
The present study establishes the characterization of HFABP test, its
association in the early diagnosis of myocardial ischemic patients and its comparison
with CK-MB, the commonly used early biochemical marker of coronary artery
disease.
In our study serum concentration of HFABP were found to be increased in
patients with coronary heart disease when compared to control group and it was
statistically significant (p value <0.01). The mean value of study and control groups
were 35.811 ± 19.2871 and 2.787 ± 1.4556. These findings are in accordance with the
study of Bhakti .N. Gami et al who reported an increased level of HFABP in CAD
than normal healthy individuals.75
84
When compared to CK-MB, HFABP showed 76.5% increase in the first 3
hours, whereas CK-MB had 58.8%. At 3 to 6 hours, HFABP showed 89.3% and CK-
MB had 85.7%. Elevated HFABP values in the early hours than CK-MB correlates
with the findings of HaticePasaoglu et al.76
Our findings also correlate with study by
McMahon et al who showed that HFABP had the sensitivity of 64.3% at 0 to 3 hours
and 85.3% at 3 to 6 hours.77
Positive correlation of HFABP and CK-MB levels with duration was also
found in this present study. This correlates with the study done by P. Mad et al.72
Uric acid levels were found to be increased in CAD patients than healthy
controls and it was statistically significant (p<0.05). Even though uric acid level was
elevated in study group than controls, it had no correlation with HFABP value in our
study (r=0.015). This finding opposses the findings of kadowaki et al who showed a
significant positive correlation of Uric acid with HFABP (r=0.166).78
Traditional risk factors used in the prediction of atherosclerosis are Total
Cholesterol, Triglycerides, LDL and HDL. Though we had significant statistical
difference between study and control group, we had no significant correlation between
HFABP with lipid profile.
In this study, there was no statistically significant difference of HFABP value
between smokers and non-smokers, alcoholics and non-alcoholics, Diabetics and non-
Diabetics. The increase of HFABP in non-Hypertensives might be due to unequal
distribution of samples. These findings did not correlate with the findings of otaki et
85
al79
who showed increased HFABP value in diabetics, Hypertensives and smokers
than non-diabetics, non-Hypertensives and non–smokers respectively.
Significant Positive correlation between HFABP and Hypertension was found
in our study (p value 0.018). This correlates with the findings of otaki et al.79
86
CONCLUSION
The ability to detect ischemia before myocyte destruction is necessary for
earlier and more accurate management decisions for the patients suspected to have
CAD. Myoglobin rises within 2 hours but it is a less specific marker. CK-MB a
commonly used early marker, lacks early sensitivity because their blood concentration
do not increase until 6 to 8 hours after onset of AMI. The other ischemic markers –
Ischemia Modified Albumin (IMA), Glycogen Phosphorylase Isoform- BB (GP-BB)
are not cardiac specific. In this study, elevated HFABP in the early hours (0 to 3
hours) of onset of ischemic chest pain clearly shows that serum HFABP can be used
as an early marker in the diagnosis of coronary artery disease. HFABP a small
molecular size protein which has absolute specificity towards myocardium, is released
in to the circulation at the early stage and presence of trace amount of HFABP in
circulation under physiological conditions implies detection of the marker in serum is
possible even with minimal increase. In conclusion, HFABP is a sensitive and specific
marker for the early diagnosis of Ischemic Heart Disease and plays a major role in the
management of Coronary Artery Disease.
87
LIMITATIONS OF THE STUDY
1. Small sample size of the study population.
2. Correlation of HFABP with Troponin-I would have helped better in assessing
the diagnostic importance of HFABP.
3. The data provided here is only one time measurement for each patient. Serial
measurement of HFABP in CAD patients would have helped in evaluating the
prognostic importance of HFABP.
4. For using HFABP as an early marker, the assay should have fast turn-around
time. The assays available to estimate HFABP in routine clinical practice is
limited because they are not automated.
5. Estimation of HFABP is cost effective.
88
FUTURE SCOPE OF THE STUDY
Estimation of HFABP as early in patients attending emergencies can be used to
rule out ischemic heart disease and also to eliminate non anginal cause of chest pain. It
can also be used as a POCT (Point Of Care Test) in the emergency department. As our
knowledge expands regarding the functions of HFABP, assessment of Serum HFABP
would be an important diagnostic marker as well as a prognostic marker for CAD. H-
FABP deserves further investigation for the early diagnosis of MI, especially in
patients presenting early after symptom onset.
89
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PROFORMA
NAME : AGE : SEX :
OP/IP.NO : DOA : DOD :
SYMPTOMS :
DURATION :
PAST HISTORY :
PERSONAL HISTORY :
EXCLUSION CRITERIA :
Symptoms of Heart Failure - Y / N
Liver Disease - Y / N
Kidney Disease - Y / N
GENERAL EXAMINATION : PR: BP:
RR:
SYSTEMIC EXAMINATION :
CVS : CNS :
RS : ABD :
INVESTIGATIONS :
ECG :
ECHO :
RANDOM BLOOD GLUCOSE :
BLOOD UREA :
SERUM CREATININE :
AST :
SERUM URIC ACID :
LIPID PROFILE :
TOTAL CHOLESTEROL :
HDL :
TGL :
LDL :
VLDL :
SERUM CK-MBLEVEL :
SERUM H-FABP LEVEL :
CONSENT FORM
“SERUM HEART TYPE FATTY ACID BINDING PROTEIN (H-FABP) AS
AN EARLY MARKER OF MYOCARDIAL ISCHEMIA”
Study Centre : Mahatma Gandhi Memorial Govt. Hospital,
Thiruchirapalli.
PATIENT NAME : AGE :
IP No : SEX :
ADDRESS :
ATTENDER'S NAME :
RELATION TO PATIENT :
The details of the study has been provided to me in writing and explained to
me in my own language. I confirm that I understood the above study.
I understand that the patient’s participation in the study is voluntary and
that I am free to withdraw from the study at any time, without giving any
reasons, without affecting the medical care that will normally be provided
by the hospital.
I understand that the doctor involved in the study does not require my
permission to assess various Biochemical parameters.
I agree not to restrict the use of any data or results that arise from this
study, provided such a use is only for scientific purpose.
I consent wholeheartedly after understanding that the study is taken up for
the benefit of the patient.
Signature/Thumb impression of the guardian
Trichy Signature of the Investigator
Date:
