LIPID PROFILE OF ENTERIC FEVER PATIENTS IN ENUGU METROPOLIS
BY
IKEGWUONU IFEOMA CHINWE
PG/MSC/02/31964
DEPARTMENT OF MEDICAL LABORATORY
SCIENCES UNIVERSITY OF NIGERIA, ENUGU CAMPUS
THIS DISSERTATION IS SUBMITTED TO THE DEPARTMENT OF MEDICAL LABORATORY SCIENCES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF MASTER OF SCIENCE (MSC) DEGREE IN MEDICAL LABORATORY SCIENCES
(CLINICAL CHEMISTRY)
FEBUARY, 2009.
1
CERTIFICATION
This is to certify that this Dissertation titled “LIPID PROFILE
OF ENTERIC FEVER PATIENTS IN ENUGU METROPOLIS”
was carried out by Ikegwuonu Ifeoma Chinwe of Department
of Medical Laboratory Sciences, University of Nigeria, Enugu
Campus under my supervision.
………………………….. ………………………….
ONYEANUSI J.C Date
Supervisor
DEDICATION
This work is dedicated to the memories of my late father,
H.R.H. Igwe Sir G.O. Achufusi, to my husband Chief Hon.
Ifenna P. Ikegwuonu and all my children Adanna, Tobenna,
Chidera, Ifeoma, Ifechukwu and Kosisochukwu.
ACKNOWLEDGEMENT
My first gratitude goes to God Almighty for the gift of
life, wisdom and strength from the beginning of this work to
its completion.
I express my sincere gratitude to my supervisor J.C.
Onyeanusi for his fatherly advice and relentless effort to see
to the completion of this work.
My special thanks goes to the Dean faculty of Health
Sciences university of Nigeria Enugu Campus Prof. N.F.
Onyemelukwe for her motherly advice and contributions to
the success of this work. I also thank my lecturers I.S.I.
Ogbu, Mr. Ureme, Mr. IG. Maduka and others for their
criticism, advice and helping out with the analysis.
This work would not have been possible without the
special contributions of the Doctors and staff of M.O.P and
G.O.P.D of both University of Nigeria Teaching Hospital and
Parklane Specialist Hospital Enugu, who made their
patients available for this project. I am also indebted to the
Doctors and Medical. Lab. Scientists of Annunciation and
Ntasi-Obi Ndi No-Na Afufu Hospitals for helping out with
their patients.
My most profound gratitude goes to my mum, children
and finally my darling husband Chief Hon. Ifenna .P.
Ikegwuonu whose love, support and caring helped me all
through these years of struggling. May God bless you all in
Jesus name Amen.
ABSTRACT
Lipid profile of three hundred subjects (two hundred positively diagnosed of
enteric fever and one hundred apparent healthy subjects) who were
attending clinics in university of Nigeria Teaching Hospital (Ituku-Ozzalla,
Parklane, Ntasi Obi-ndi-no-na-afufu and Annunciation, Hospitals Enugu
were estimated. The enteric fever was investigated using slide and tube
agglutination method and confirmed using enterocheck WB kit from Zephyr
Biomedicals Verna, India. The serum cholesterol (TC), Triglycerides (TG) and
High density Lipoprotein cholesterol (HDL-C) were assayed using enzymatic
method while very low density lipoprotein cholesterol (VLDL-C) and low
density lipoprotein (LDL-C) levels were estimated using Friedewald formula.
The ages of all subjects were between twenty and forty years. The results
showed no significant difference (P>0.05) in the mean values of TC, TG,
HDL-C, VLDL-C and LDL-C for patients and controls. However, there was
statistically significant difference (P<0.05) in TC (4.74+ 1.56, 4.22 + 0.92)
and LDL-C (2.87 + 1.34, 2.42+ 0.71) of male patients and controls
respectively. Also in female patients and controls there was statistically
significant difference (P<0.05) in TC (4.41 + 0.75, 4.77 + 0.89) and LDL –C
(2.62+ 0.74, 2.90+ 0.86) respectively. There was a correlation between TG,
VLDL-C, HDL-C, LDL-C and ages of patients and controls. TG (r= 0.67,
P<0.001, r = 0.79, P<0.001), VLDL- C (r = 0.67, P<0.0001, r= 0.79, P<0.001),
HDL –C (r = -0.27,P= 0.0001, r = -0.45, P<0.001), LDL –C(r=-0.18, p= 0.117,
r = -0.40, P<0.001) respectively. TC and LDL –C were found to be gender-
dependent while TG, TC, HDL –C, LDL – C and VLDL –C were all found to be
age –influenced. Again this study suggested that lipid profile do not alter in
patients with enteric fever.
LIST OF TABLES
TABLE TITLE PAGE
2:1 Physical and chemical description of plasma
lipoproteins in humans ……………………..…………19
2.2 Classification and properties of major human plasma
apolipoproteins…………………...……………………….22
2.3 Physiological functions of the apolipoproteins in
human
plasma…………..…………….…………….……26
4.1 Lipid profile of enteric fever patients and of
controls….………….…………..…………………………..62
4.2 Lipid profile levels of male patients and age –
matched controls..
…….…………………………………63
4.3 Lipid profile of female patients and age-matched
controls…..…………
………………………………………64
4.4 Lipid profile levels of patient (M & F) and age-
matched controls (M&F) grouped according to age
(in fives)………………………………………………………65
4.5 Correlation of parameters (TG, TC, HDL-C, LDL-
C, VLDL-C) with age and tire O and H of
patients...66
LIST OF FIGURES
FIGURES TITLE
PAGE
4.1 Relationship between serum Triglycerides of patients and
age………………………………………………………………….............6
6i
4.2 Relationship between serum total cholesterol of patients and
age………………………………….………………………..………………6
6ii
4.3 Relationship between serum HDL – Cholesterol of patients
and
age……………………………………..……………………………....66iii
4.4 Relationship between serum LDL – cholesterol of patients
and
age…………………………………………………………………..….66iv
4.5 Relationship between serum VLDL – Cholesterol of patients
and
age……………………..……..…………………………………..……66v
4.6 Relationship between serum triglycerides of controls and
age…………………………….…………………………………..…..…….6
6vi
4.7 Relation betweens serum HDL-cholesterol of controls and
age…………………………………………………………………..………66
vii
4.8 Relationship between serum HDL – cholesterol of
controls and
age…………………….……..……….………………….66viii
4.9 Relationship between serum LDL – cholesterol of
controls and age
………………….……..………….………………....66ix
4.10 Relationship between serum VLDL – cholesterol of
controls and age
……………………………………………………..…66x
4.11 Relationship between lipids profile of patients and age groups
(in
five)…………………………………………………………………………66
xi
4.12 Relationship between lipids profile of controls and age
groups (in
five)………………………………………...……………….66xii
LIST OF ABBREVIATIONS
Abbrev. Full Meaning
HDL High Density Lipoprotein
LDL Low Density Lipoprotein
VLDL Very Low Density Lipoprotein
TG Triglyceride
TC Total serum Cholesterol
FFA Fatty acids
LCAT Lecithin Cholesterol Acyl Transferase
IDL Intermediate Density Lipoprotein
LP (a) Lipoprotein (a)
Apo Apolipoprotein
SD Standard Deviation
Ig Immunoglobulin
HTGL Hepatic Triglyceride Lipase
LDL Lipoprotein Lipase
CAD Coronary Artery Disease
DNA Deoxyribonucleic Acid
DM Diabetes Mellitus
CETP Cholesteryl Ester Transfer Protein
MP Malaria Parasite
ML Milliliter
MMOL/L Milimole Per Liter
NS Not Significant
SG Significant
FH Familial Hypercholesterolemia
TABLE OF CONTENTS
Approval page…………………………………………………………i
Dedication………………………………………………………………ii
Acknowledgement..…………………………………………………..iii
Abstract…………………………………………………………………iv
List of Tables……………………………………………………………v
List of Figures………………………………………………………….vi
List of Abbreviations…………………………………………………vii
Table of Contents…………………………………………………….viii
CHAPTER ONE: INTRODUCTION
1.1 Background of the study….…………………………….…….1
1.2 Aim and Objectives ……………………………………5
CHAPTER TWO: LITERATURE REVIEW
2.1Pathphysiology of Enteric Fever……………………….…….6
2.2 Pathogenesis of Enteric Fever…………….…………….……6
2.3 Lipid Chemistry……….…………………………………….…..8
2.4 Classification of Lipids…….…………………………………..8
2.5 Properties of Lipids…….………………………………………11
2.6 Lipoproteins…………………..…………………………………13
2.6.1 Functions of Lipoproteins………………………………….14
2.6.2 Classification of Lipoproteins……………………………..15
2.7 Apolipoproteins…………………….………………………….20
2.8 Factors Affecting Serum Lipid Levels…...….……..…......33
2.9 Lipid Metabolism in Diseases………………………..........36
2.10 Lipid Metabolism in Enteric Fever…………………………44
2.11 Normal Expected Lipids values……..……………………...45
CHAPTER THREE: MATERIALS AND METHODS.
3.1 Subjects………………………………..………………………….49
3.2 Samples.…………………………………………………………..49
3.3 Methods for Enteric Fever Estimation……..………………50
3.4 Enteric fever Confirmatory Test……………..………………52
3.5 Methods for Lipid Profile Estimation……………….………54
CHAPTER FOUR: RESULTS……………………………………..59
CHAPTER FIVE: DISCUSSION AND CONCLUSION……….67
REFERENCES………………………………………………………..70
APPENDIX 1…………………………………………………………..76
APPENDIX 11…………………………………………………………89
APPENDIX111…………………………………………………………91
CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
Enteric fever or typhoid fever is a major public health problem
in the developing countries of the world with an estimated annual
incidence of 540 per 100,000 (Katung, 2000). Typhoid fever is endemic
in the economically disadvantaged countries in Africa like Nigeria,
Asia and South and Central America (Philip 2000).
Enteric fever is a severe bacterial infection which is used to
describe two different but similar diseases known as typhoid fever and
paratyphoid fever. Typhoid fever is caused by Salmonella typhi while
paratyphoid fever is caused by Salmonella paratyphi A, Salmonella
paratyphi B and Salmonella paratyphi C. Salmonella typhi must be
ingested to cause disease. Transmission often occurs when a person
in the carrier state does not wash hands thoroughly (or not at all) after
defecation and serves food to others. This pathway is sometimes
called the fecal-oral route of disease transmission. In countries where
open sewage is accessible to flies, the insects land on the sewage, pick
up the bacteria, and then contaminate food to be eaten by humane.
After being swallowed, the S.typhi bacteria head down the digestive
tract, where they are taken in by cells called mononuclear phagocytes.
These phagocytes are cells of the immure system, whose job it is to
engulf and kill invading bacteria and viruses. In the case of S.typhi,
however the bacteria are able to survive ingestion by the phagocytes
and multiply within these cells. This period of time, during which the
bacteria are multiplying within the phagocytes, is the 10 to 14day
incubation period of typhoid fever. When huge numbers of bacteria fill
an individual phagocyte, they spill out of the cell and into the
bloodstream, where their presence begins to cause symptoms. The
presence of increasingly large numbers of bacteria in the bloodstream
(bacteremia) is responsible for an increasingly high fever, which lasts
throughout the four to eight weeks of the disease in untreated
individuals. Other symptoms of typhoid fever include constipation (at
first), extreme fatigue, headache, joint pain, and a rash across the
abdomen known as rose spots. (Encyclopedia of medicine, 2002).
The bacteria move from the bloodstream into certain tissues of
the body, including the gallbladder and lymph tissue of the intestine
(called peyer’s patches). The tissue’s response to this invasion causes
symptoms ranging from inflammation of the gallbladder (cholecystitis)
to intestinal bleeding to actual perforation of the intestine. Perforation
of the intestine refers to an actual hole occurring in the wall of the
intestine, with leakage of intestinal contents into the abdominal
cavity. This leakage causes severe irritation and inflammation of the
lining of the abdominal cavity, which is called peritonitis. Peritonitis
is a frequent cause of death from typhoid fever (Encyclopedia of
Medicine, 2002).
Other complications of typhoid fever include liver and spleen
enlargement, sometimes so great that the spleen ruptures or bursts;
anemia, or low red blood cell count due to blood loss from the
2
intestinal bleedings; joint infections, which are especially common in
patients with sickle cell anemia and immune system disorders,
pneumonia caused by a bacterial infection-usually streptococcus
pneumoniae which is able to take hold due to the patient’s weakened
state; heart infections and meningitis and infections of the brain,
which cause mental confusion and even coma. It may take a patient
several months to recover fully from untreated typhoid fever.
All these complications of typhoid fever lead to severe sepsis and
lipid profile is known to alter in patients with severe sepsis (Khosla,
1991).
Lipid profile is a group of blood tests that tells how the body
uses, changes or stores lipids. Lipids synthesized in the liver and the
intestine have to be transported to the various tissues to accomplish
their metabolic functions. Because of their insolubility, they are
transported in the plasma in macromolecular complexes called
lipoproteins. Lipoproteins are spherical particles with nonpolar
lipids(triglycerides and cholesterol esters) in their core and more polar
lipids(phospholipids and free cholesterol) oriented near the surface.
The amount of lipoproteins in the blood can change with what you eat,
with some illness and because of heredity. (Ohio Health, 2004). Some
of the lipids done in the profile are cholesterol and triglycerides.
Cholesterol is found almost exclusively in animals,in which it is
also the main sterol. These cholesterol is used by the body to help
build cells and produce certain hormones and bile salts. Cholesterol
forms complexes with protein in the blood to produce lipoproteins.
3
Lipoproteins come in two forms (1) High Density lipoproteins (HDL),
the good cholesterol with more protein than fat and (2) Low Density
lipoprotein (LDL) which is the bad cholesterol with more fat than
protein (Ohio Health, 2004). These cholesterols are mostly produced
by the liver and derived from foods eaten. The normal range of
cholesterol in the blood should be less than 200 milligrams per
deciliter or mg/dl. High cholesterol of 240mg/dl or greater in the
blood increases the risk of heart disease, stroke, coronary artery
disease etc. Abnormally low levels of cholesterol may indicate
hyperthyroidism or an overactive thyroid gland, liver disease,
inadequate absorption of nutrients from the intestines and
malnutrition (Ohio health, 2004).
Triglycerides (Triacylglycerol) are the principal form of fats,
circulating in the blood stream. (Ethnomed Org, 2004). Most of the
body fat comes in this form.
Triglycerides are derived from two sources namely:
(a) From the foods we eat, mainly sugar, animal products and
saturated fats.
(b) From the liver itself (Ethnomed.Org, 2004).
4
1.2 Aims and Objective
(1) To determine the lipid profile of patients with enteric/typhoid fever
and control.
(2) To compare the mean values of lipids under study in both
controls and enteric fever patients.
(3) To compare changes in lipid profile seen in enteric fever caused
by different salmonella species.
(4) To ascertain whether changes in the lipid profiles are gender
dependent.
5
CHAPTER TWO
LITERATURE REVIEW
2.1 Pathophysiology of Enteric Fever
Following ingestion of an infectious dose of at least 10,000
bacteria, mucosal penetration occurs in the distal ileum resulting in a
transient asymptomatic bacteria. The organisms survive and multiple
within mononuclear phagocytes located in peyer patches of the ileum,
lymph nodes, spleen, liver and bone marrow. The clinical phase of the
disease begins within 1-3 weeks, resulting from persistent bacteremia.
Hematogenous spread to ileal peyer patches and the gall bladder
reintroduces bacteria to the gut lumen and stool culture becomes
positive, allowing continued fecal oral spread of the disease. Mucosal
ulceration overlying hyperplastic peyer patches in the ileocecal region
may result in pain, diarrhea, bleeding and occasional perforation
(katung, 2000)
2.2 Pathogenesis of Enteric Fever
The bacteria enter the human digestive tract, penetrate the
intestinal mucosa (causing no lesion), and are stopped in the
mesenteric lymph nodes. There, bacterial multiplication occurs, and
part of the bacterial population lyses. From the mesenteric lymph
nodes, viable bacteria and lypopolysaccharide (endotoxin) may be
released into the bloodstream resulting in septicemia. Release of
endotoxin is responsible for cardiovascular “collapses and tuphos” (a
6
stuporous state-origin of the name typhoid) due to action on the
ventriculous neurovegetative centers.
Salmonela excretion by human patients may continue long after
clinical cure. Asymptomatic carriers are potentially dangerous when
unnoticed. About 5% of patients clinically cured from typhoid remain
carriers for months or even years. Antibiotics are usually ineffective on
salmonella carriage (even if salmonellae are susceptible to them)
because the site of carriage may not allow penetration by the
antibiotic.
Salmonellae survive sewage treatments if suitable germicides
are not used in sewage processing. In a typical cycle of typhoid,
sewage from a community is directed to a sewage plant. Effluent from
the sewage plant passes into a coastal river where edible shellfish
(muscles, oysters) live. Shellfish concentrate bacteria as they filter
several liters of water per hour. Ingestion by humans of these seafoods
(uncooked or superficially cooked) may cause typhoid or other
salmonellosis.
Salmonellae do not colonize or multiply in contaminated shellfish
(Thielman, 2005).
Typhoid is strictly a human disease. The incidence of human
disease decreases when the level of development of a country
increases (i.e, controlled water sewage systems, pasteurization of milk
and dairy products). Where these hygienic conditions are missing, the
probability of fecal contamination of water and food remains high and
so is the incidence of typhoid.(Thielman,2005).
7
2.3 Lipid Chemistry
Lipids are ubiquitous in the body tissues and have an important
role in virtually all aspects of life serving as hormones or hormone
precursors, aiding in digestion, providing energy storage and
metabolic fuels, acting as functional and structural components in cell
membranes, and forming insulation to allow nerve conduction or to
prevent heat loss. However, only a limited number of the numerous
different lipids known to exist in humans are usually of clinical
importance (Burtis and Ashwood 1999).
2.4 Classification of Lipids
2.4.1 Lipids are classified as simple or complex. simple lipids:
These are esters of fatty acids with various alcohols. They
include
(a) Neutral fats:- which are esters of fatty acid and glycerol
(triglycerides) e.g Tripalmitic and stearin.
(b) Waxes:- Which are esters of fatty acids with higher
molecular weight monohydric alcohols e.g beewax, sperm
oil, wool wax and carnauba wax.
2.4.2. Complex Lipids:- These are esters of fatty acids
containing groups in addition to an alcohol and a fatty acid.
(a) Phospholipids:- These are lipids containing, in addition to fatty
acids and an alcohol, a phosphoric acid residue. They frequently
have nitrogen-containing bases and other substituents, e.g in
8
glycerophospholipids the alcohol is glycerol and in sphingo-
phospholipids the alcohol is sphingosine.
(b) Glycolipids (glycosphingolipids). These are lipids containing a
fatty acid, sphingosine, and carbohydrate, e.g
bi) Cerebrosides:- These contain galatose or glucose, a high
molecular weight fatty acid and sphingosine. Structurally,
they are similar to sphingomyelin e.g kersins, cerebrone,
nervons, oxynervons. They are found mainly in brain tissue and
in other tissues such as reticuloendithelial cell and nerve fibres
especially in myelinated nerve fibres.
bii) Sulphatides:- Sulphatides are sulphate derivatives of the
galactosyl residue in cerebrosides.
biii) Gangliosides:- These are glycolipids occurring in the brain (in
ganglionic cells). The main components are sphingosine, fatty
acid and branched chain carbohydrates with as many as seven
sugar residues.
C. Other Complex Lipids: Lipids such as sulfolipids and
aminolipids. Lipoproteins may also be placed in this category.
2.4.3. Precursor and derived lipids: These include fatty acids,
glycerol, steroids, other alcohols, fatty aldehydes, and ketone
bodies, hydrocarbons, lipid- soluble vitamins and hormones.
The acylglycerols (glycerides), cholesterol, and cholesteryl
esters are termed neutral lipids because they are uncharged.
9
2.4.4 Fatty acids: Fatty acids occur mainly as esters in natural fats
and oils but do occur in the unesterified form as free fatty acids, a
transport form found in the plasma. They are aliphatic (straight
chain) substances, which have at least carbon atoms and a terminal
carboxylic acid (C00H) group.
The general formula may be given as R. C00H, where R is the
non carboxylic acid radical of the molecule. For monocarboxylic acids
this may be taken as CnH2+1. In human metabolism the most
important fatty acids are palmitic and stearic acids, which are both
saturated (have no double bond) and conform to the general formula.
Palmitic acid (n=16) C6H33C00H
Stearic acid (n=18) C18H37C00H
Fatty acids not bound to other substances are termed free fatty
acids (FFA) or non-esterified fatty acid. More commonly, however they
are combined with glycerol to form glycerides.
2.4.5 Triglycerides: Most fatty acids form esters known as glycerides
or glycerol, of which triglycerides are the most abundant.
Triglycerides are lipids of natural fats consisting of glycerol combined
with three fatty acid molecules (concise medical Dictionary, 1998).
O II H2C1-0-C-R1
O
II R2-C-0-C1H
O II CH2-0-C-R2.
10
Triglycerides are the major components of most foods, typically
making up more than 65% of total lipids present (Winkleman and
Wybenega, 1974). Triglycerides are the main storage forms of fatty
acids. Triacylglycerols are ester of the trihydric alcohol glycerol and
fatty acids. Mono-and di-acylglycerols wherein one or two fatty acids
are esterified with glycerol are also found in the tissues. These are of
particular significance in the synthesis and hydrolysis of
triacylglycerols.
2.5 Properties of Lipids
2.5.1 Physical Properties
a) Solubility: Lipids exhibit a very low solubility in water because
of the essentially hydrocarbon nature of the common fatty acids.
(Burtis and Ashwood, 1999). The low molecular weight fatty acids –
butyric acids are miscible with water, whereas fatty acids having more
than six carbon atoms as in caproic acid are essentially insoluble in
water but are soluble in nonpolar solvents (Burtis and Ashwood,
1999).
Some phospholipids (polar lipids) also contain a large proportion
of polar groups and are therefore partly soluble in water and partly
soluble in nonpolarsolvent. (Delvin, 1993). The molecules thus become
oriented at oil-water interphase with the polar group in the water
phase (Delvin, 1993). A critical concentration of polar lipids is present
in an aqueous medium and they form what is called micelles. Non-
polar lipids cannot form micelles, but they can be incorporated into
the non-polar interior of the micelles in the form of mixed micelle.
11
b) Melting Point: In triacylglycerol both a decrease in chain length
and more importantly an increase in the degree of unsaturation
of the hydrocarbon will lower its Melting point. This is a
reflection of its complete fatty acid composition (Delvin, 1993).
2.5.2 Chemical Properties
A) Hydrolysis: Lipids such as triacylglycerol (neutral fat) may be
enzymatically hydrolyzed to free fatty acids and glycerol. This
hydrolysis may also be accomplished by an alkali. The alkali-
catalyzed hydrolysis of a lipid is called saponification and it yields the
alkali salt or soaps of fatty acids and glycerol (Murray et al 2000).
Triacylglycerol KOH Glycerol + Alkali salt of the fatty acid (soap)
B) Addition Reaction: Hydrogenation and Halogenation.
Hydrogenation of unsaturated fats in the presence of a catalyst
(e.g. nickel) is known as Hardening. The industrial process is
commercially valuable as a method of converting this liquid fat
usually of plant origin into solid fats like margarine (Delvin,
1993).
C) Rancidity: This is a change that results in unpleasant odour
and taste in a fat. The oxygen of the air attacks the double bond
in fatty acids to form a peroxide linkage. Free radicals (Highly
reactive) are produced leading to chain reaction. Lead or copper
catalyses rancidity; while exclusion of oxygen or the addition of
an antioxidant delays the process. Living tissues unless
antioxidants such as tocopherol (vitamin E) are present to
12
scavenge the free radicals formed. Peroxidation is also catalyzed
invivo by heme compounds and by the enzyme lipoxygenase
found in platelets (Delvin, 1993)
D) Spontaneous Oxidation: Oil that contain highly unsaturated
fatty acids (e.g linseed oil) are spontaneously oxidized by
atmospheric oxygen at ordinary temperature to form a hard
proof. They are therefore known as drying oil.
2.6 Lipoprotein
A lipoprotein is a biochemical assembly that contain both
proteins and lips. The lipids or their derivatives may be covalently or
non-covalently bound to the proteins. Many enzymes, transporters,
structural proteins, antigens, adhesions and toxins are lipoproteins.
Examples include the high density and low density lipoproteins of the
blood, the transmembrane proteins of the mitochondrion and the
chloroplast, and bacterial lipoprotein (Torellin, 2005).
Lipids synthesized in the liver and the intestine have to be
transported to the various tissues to accomplish their metabolic
functions. Because of their insolubility, the lipids are transported in
the plasma in macromolecular complexes called lipoproteins.
Lipoproteins are spherical particles with nonpolar lipids
(triglycerides and cholesterol esters) in their core and more polar lipids
(phospholipids and free cholesterol) oriented near the surface. They
also contain one or more specific proteins, called apolipoproteins, that
are located on their surfaces. The association of the core lipids with
13
the phospholipid and protein coat is noncovalent, occurring primarily
through hydrogen bonding and vander waals forces. This binding of
lipid to protein is loose enough to allow the ready exchange of lipids
among the plasma lipoproteins and between cell membrane and
lipoprotein, yet strong enough to allow the various classes and
subclasses of lipoprotein to be isolated by a variety of analytical
techniques.
2.6.1 Functions of Lipoprotein
The lipids are often an essential part of the complex even if they seem
to have no catalytic activity themselves. To isolate transmembrane
lipoproteins from their associated membranes, detergents are often
needed. All cells use and rely on fats and for all animal cells,
cholesterol as building blocks to create the multiple membranes which
cells use to both control internal water content, internal water soluble
elements and to organize their internal structure and protein
enzymatic systems.
Lipoproteins in the blood, a water medium, carry fats around
the body. The protein particles have charged groups aimed outward so
as to attract water molecules, this makes them soluble in the salt
water base blood pool. Triglyceride fats and cholesterol are carried
internally, shielded by the protein particle from the water. The
interaction of the proteins forming the surface of the particles with (a)
enzymes in the blood, (b) with each other and (c ) with specific
proteins on the surfaces of cells determine whether triglycerides and
14
cholesterol will be added to or removed from the lipoprotein transport
particles. (Torelli, 2005).
2.6.2 Classification Of Lipoproteins
The classification of lipoproteins depends a great deal on the
method used for analysis (Gambino,1986). Several analytical systems
have been used to isolate, separate and characterize lipoproteins,
most of which are based on one or another physiochemical property of
the lipid-protein complex. The four most frequently used systems are
based on analytical ultracentrifugation, preparative
ultracentrifugation, electrophoresis and precipitation techniques
(Gambino, 1986, Natio, 1989b). With a paper or agarose support
medium, electrophoresis patterns show that chylomicrons remain at
the origin, while pre-beta-lipoproteins and beta-lipoproteins migrate in
the beta1 and beta2-globulin areas respectively, and alpha-lipoproteins
migrate in the alpha1-globulin area.
Using the ultracentrifuge and taking advantage of the fact that
lipoproteins are lighter than the other serum proteins, one can
separate the lipoproteins into chylomicrons, very low-density
lipoprotein (VLDL), low density lipoprotein (LDL), and high density
lipoprotein(HDL). These lipoprotein classes correlate with
electrophoresis patterns, for example, pre-beta-lipoprptein is generally
synonymous with VLDL, beta-lipoprotein with LDL, and alpha-
lipoprotein with HDL.
15
2.6.2.1 Chylomicrons
Chylomicron is the term originally used to describe the
microscopically visible particle appearing in the plasma after a fatty
meal. Subsequently,it has been characterized as triglyceride-rich
particle secreted by the intestine, which serves as the major transport
form of dietary fat. It is the lightest lipoprotein of a density less than
plasma, and contains triglyceride combined with cholesterol, small
amounts of phospholipids, and specific apoproteins (Apo B, Apo A,
Apo C, and Apo E). Under fasting conditions (more than 12 hours after
meal), no chylomicrons are generally found in the blood (Natio,
1989b).
2.6.2.2 VLDL
An average preparation of VLDL contains 52% triglyceride, 18%
protein. Cholesterol and cholesteryl esters occur in a ratio of 1:1 by
weight. Sphingomyelin and phosphatidyl choline are the major
phospholipids. Apo B appears to be present in a constant absolute
quantity in all VLDL fractions, and account for approximately 30-35%,
with Apo C making up over 50% of the apoprotein content in VLDL.
Apo E and varying quantity of other apoproteins may also be present.
The relative quantity of each protein varies with the individual and
with the degree of hyperlipidaemia. On ultracentrifugation, VLDL
separates at density below 1.006g/ml (after chylomicron removal).
16
2.6.2.3. LDL.
This separates at densities between 1.006 and 1.063g/ml on
ultracentrifugation. It contains, by weight, 80% lipid and 20% protein,
and is smaller in size (21-25nm). About 60% of LDL lipid is
cholesterol. LDL constitutes 40%-60% of the plasma lipoprotein mass
in humans, and is the major carrier of cholesterol. Apo B is the major
apoprotein of LDL, and LDL Apo B represents 90-95% of the total
plasma Apo B. LDL is frequently separated into two classes,
LDL1(intermediate density lipoprotein, IDL) and LDL2, on the basis of
floatation density. The lower density fraction, IDL (1.006-1.019g/ml) is
more lipid rich than LDL2 (1.019-1.063g/ml), and probably represents
an intermediate in VLDL catabolism. Thus, a comparison of IDL with
LDL2 demostrates the gradual disappearance of triglyceride and
apoproteins more characteristic of VLDL (Apo C and Apo E) and an
enrichment with Apo B and cholesterol ester.
2.6.2.4. HDL
HDL is the smallest of the lipoproteins (9-12nm in diameter)
and floats at the highest density (1.063-1.21g/ml) of any of the
lipoprptein molecules. The HDL macro-molecular complex contains
approximately 50% protein and 50% lipid. The quantitatively most
important HDL lipid is phospholipids although HDL cholesterol is of
particular interest. The major phospholipids species is phosphatidyl
choline(lecithin), which accounts for 70-80% of the total
phospholipids. It has an important role as a reactant in plasma
17
cholesterol esterification, which is catalysed by the enzyme
lecithin:cholesterol acyl transferase(LCAT).
On differential ultracentrifugation, HDL may be further
subfractioned into HDL2 (with a density of 1.063-1.110g/ml) and
HDL3 (1.110-1.21g/ml). HDL2 is present in pre-menopausal women at
about three times its concentration in men. Persons with lower HDL2
levels are apparently more susceptible to premature coronary heart
disease (CHD) (Naito,1989b)
2.6.2.5. Other Lipoproteins.
a) Floating beta-lipoprotein, or beta migrating VLDL. This type of
lipoprotein is found in persons with type111
hyperlipoproteinaemia or broad-beta disease(derived from the
broad smear from beta to pre-beta-lipoprotein regions frequently
present on whole plasma lipoprotein electrophoresis in these
subjects). This fraction has a density of 1.006g/ml, which is
characteristic of VLDL, but has a beta-lipoprotein migration
pattern. The abnormal lipid composition of VLDL in type111
hyperlipoproteinaemic persons is attributable to a
proportionately larger amount of cholesterol in that fraction.
b) Lp(a) or sinking pre-beta lipoprotein.
This resembles LDL in lipid composition, concentration and
density(1.005-1.010g/ml), but is differentiated by
immunological tests. 65% of the Lp(a) protein is Apo B, 15% is
18
albumin, and the remainder is an apoprotein unique to Lp(a),
called Apo Lp(a).
(c) Lipoprotein X.
This is found most characteristically in plasma of patients with
biliary obstruction. It resembles LDL in floatation density, but
has different lipid and protein composition, electrophoresis
mobility (Naito,1989b).
Table2:1;
Physical and Chemical description of plasma lipoprotein in
humans (Naito,1989b)
Feature Chylomicron VLDL IDL LDL HDL
Density (glml) <1.006 <1.006 1.006-
1.019
`1.019-
1.063
1.063-
1.21 Electrophonesis Mobility Origin Pre-beta Beta Beta Alpha
Floatation rate (Sf) >400 20-400 12-20 0-10 -
Diameter (nm) 80-500 40-80 24.5 20 7.5-12
Lipids (%by weight) 98 92 85 79 50
Cholesterol 9 22 35 47 19
Triglyceride 82 52 20 9 3
Phospholipids 7 18 20 23 28
Apoproteins (%by weight major)
2 8 15 21 50
A-1, A-11 - - - A-1,
A-11 B B B B -
C-1, C-11 C-111
C-1, C-11
C-111
-
-
-
E E E - -
19
Minor: - A-1, A-11
- C-1, C-11
C-111
C-1, C-11
C-111 - - - D
- - - E
2.7 Apolipoproteins
Apolipoproteins- They are lipid-binding proteins which are the
constituents of the plasma lipoproteins, sub-microscopic spherical
particles that transport dietary lipids through the bloodstream from
the intestine to the liver and endogenously synthesized lipids from the
liver to tissues that can store them (adipocytes), metabolize them
(muscle, heart, lung) or secrete them(breast).
The amphipathic (detergent-like) properties of apolipoproteins
solubilize the hydrophobic lipid constituents of lipoproteins, but
apolipoproteins also serve as enzyme co-factors, receptor ligands, and
lipid transfer carriers that regulate the intravascular metabolism of
lipoproteins and their ultimate tissue uptake. (Wikimedia foundation,
2007.
2.7.1 Classes of Apolipoproteins
There are five major classes of apolipoproteins, and several sub-
classes.
(a) Apolipoprotein A (apo A-1, apo A-11, apo A-iv and apo A-v)
(b) Apolipoprotein B (apo B48 and apo B100).
(c ) Apolipoprotein C (apo C-1, apo C-11, apo C-111, and apo C-iv)
20
(d) Apolipoprotein D
(e) Apolipoprotein E
Hundreds of genetic polymorphisms of the apolipoproteins have been
described, and many of them alter their structure and function.
Synthesis and Regulation
Apolipoprotein synthesis in the intestine is regulated principally by
the fat content of the diet. Apolipoprotein synthesis in the liver is
controlled by a host of factors, including dietary composition,
hormones (insulin, glucagons, thyroxin estrogens, androgens), alcohol
intake, and various drugs (statins, niacin, and fibric acids).
21
Table2:2
Classification and Properties of Major Human Plasma
Apolipoproteins
APOLIPO
PROTEIN
MOLECULAR
WEIGHT
(DATTONS)
CHROMOSOMAL
LOCATION
FUNCTION LIPOPROTEIN
CARRIERS
Apo A-1 29016 11 Cofactor LCAT chylomicron HDL
Apo A-11 17414 1 Not known HDL
Apo A-iv 44, 465 11 Activates
LCAT
CHYLOMICRON,
HDL
Apo B-100 512 723 2 Secretion of
triglyceride
from liver
binding
protein to LDL
receptor
LDL
APO B-48 240 800 2 secretion of
triglyceride
from intestine
Chylomicron
Apo C-1 6630 19 Activates
LCAT (?)
Chylomicron,
VLDL, HDL
Apo C-11 8900 19 Cofactor LPL Chylomicron,
VLDL, HDL
Apo C- III 8800 11 Inhibits Apo
C-II activation
of LPL
Chylomicron,
VLDL, HDL
Apo E 34145 19 Facilitates
uptake of
chylomicron
reminant and
IDL
Chylomicron,
VLDL, HDL
Apo (a) 187000-
662 000
6 Unknown LP(a)
22
2.7.1 Apolipoprotein A – Apolipoprotein A-1 and apo A-II constitute
about 90% of total HDL protein. The ratio of apo A-I to A-II in HDL is
about 3:1. In addition to being an important structural component of
HDL, apo A-I is a cofactor for LCAT, the enzyme responsible for
forming cholesteryl esters in plasma. Some evidence suggests that apo
A-II may inhibit LCAT and activate hepatic triglyceride lipase. (Burtis
and Ashwood, 1999). Apo A-iv is a component of newly secreted
chylomicrons, but is not a major constituent of chylomicron
remnants, VLDL, LDL, and HDL. The primary function of apo A-iv is
currently unknown, but it has been shown to activate LCAT in vitro,
and available data suggest it plays a role in the transport of
cholesterol from peripheral tissues to the liver (burtis and Ashwood,
1999).
2.7.1.2 Apolipoprotein B- Apolipoprotein B exists in two forms: apo
B-100 and apo B-48. The two proteins are known to be translation
products of a single structural gene. Apo B-100, a single polypeptide
of over 4500 amino acids, is the full-length translation product of the
apo B gene. In humans, apo B-100 is made in the liver and secreted
into plasma as part of VLDL. Apo B-100 is the major apolipoprotein of
LDL, the end product of VLDL catabolism.
Each VLDL particles contains one molecule of apo B-100. In the
fasting state, most of the apo B in plasma is apo B-100. Unlike the
other apolipoproteins, however, apo B-100 cannot move from one
lipoprotein particle to another, and VLDL apo B-100 remains with the
23
lipoprotein as it is catabolized to LDL. Apo B-48 contains 2152 amino
acids and is identical to the amino-terminal portion of apo B-100.
Apo B-48 results from the posttranscriptional modification of internal
apo B-100 mRNA, in which a single base substitution produces a stop
codon corresponding to residue 2153 of apo B-100. Apo B-48 is made
in the intestine and is the major apo B component of chylomicrons.
Both apo B-100 and B-48 play important roles in the secretion of
VLDL and chylomicrons, respectively. Apo B-100 is recognized by the
LDL receptor in hepatic and peripheral tissues and allows the LDL
receptor-mediated internalization of LDL (Burtis and Ashwood, 1999).
2.7.1.3 Apolipoprotein C- Apolipoprotein C-I , C-II and C-III are
associated with all lipoproteins except LDL. Apo C-I the smallest of the
C apolipoproteins, has been reported to activate LCAT in vitro. Apo C-
II plays an important role in the metabolism of triglycerides-rich
lipoprotein (VLDL and chylomicrons) by activating lipoprotein lipase
(LPL), an enzyme that hydrolyzes lipoprotein triglycerides. Because of
differences in sialic acid content, apo C-111 exists in at least three
polymorphic forms. The precise metabolic function of apo C-II1 is
unknown, but it may inhibit LPL and activate LCAT, and therefore
may regulate the activities of these enzymes. (Burtis and Ashwood,
1999).
2.7.1.4 APOLIPOPROTEIN E- Apolipoprotein E is a 34-K Dal plasma
glycoprotein that is found primarily in chylomicrons, VLDL, HDL, and
chylomicron and VLDL remnants. Removal of apo E- bearing
24
lipoprotein is mediated by several different cellular receptors that
recognize a cluster of positively charged amino acids in a specific
region of apo E. Apo E plays a central role in the metabolism of
chylomicrons and VLDL remnants. It regulates and facilitates
lipoprotein uptake in the liver through;
(i) Interaction of chylomicron remnants with chylomicron remnant
receptors.
(ii) Binding of VLDL remnants to the LDL (B, E) receptor. (Burtis
and Ashwood, 1999).
There are three common apo E variants designated E2, E3 and
E4, which were initially distinguished by isoelectric focusing. These
isoforms have amino acid substitutions at residues 112 and 158.
Apo E2 has cysteine residues in both positions and apo E4 has
arginine residues in both positions, whereas apo E3 has cysteine and
arginine at positions 112 and 158, respectively. Apo E2 exhibits
reduced binding affinity for the B/E remnant receptor compared with
apo E3 which can lead to an accumulation of apo E-containing
lipoproteins in the circulation, whereas apo E4 containing lipoproteins
are cleared more rapidly than those containing apo E3. These isoforms
are coded for by the three alleles of the apo E gene, E2, E3, and E4. The
E3 alleles is most frequent, although the relative proportions of the
three alleles vary among populations. These apo E alleles have been
shown to contribute significantly to the variability of LDL cholesterol
and apo B-100 levels within populations. People with at least one E2
allele tend to have lower levels of apo B-100 and LDL cholesterol than
25
do those who are homozygous for the E3 allele, where people with at
least one E4 allele tend to have higher levels.
2.7.2 Functions of Apolipoprotein
Apolipoproteins collectively have three major physiological
functions. They are involved in
(1) Activating important enzymes in the lipoproteins metabolic
pathways.
(2) Maintaining the structural integrity of the lipoprotein complex.
(3) Facilitating the uptake of lipoprotein into cells through their
recognition by specific cell surface receptors.
Table2:3 Physiological Functions of the Apolipoproteins in
Human Plasma FUNCTION APOLIPOPROTEIN
Cofactor for enzyme Lipoprotein lipase C- II
Lecithin: Cholesterol acyltransferase A-I
Ligand on lipoprotein particle for interaction with
receptor site on cells
Remnant Receptor E
LDL receptor B-100, E
HDL receptor A-1
Structural protein on lipoprotein particle
Intestinal chylomicron
B-48, B-100
Hepatogenous VLDL B-100
HDL A-I
26
During the last decade, several physiological functions have
been identified for the apolipoproteins in plasma (as shown above).
Apo B-100 and B-48 are required as structural constituents of
lipoproteins particles for the secretion of triglyceride-rich lipoproteins
from the intestine and liver. Defects in the structure of apo B or in the
assembly of apo B-containing lipoproteins result in the failure of
intestinal and hepatogenous triglyceride-rich lipoproteins to be
secreted. Patients with this type of dyslipoproteinemia have
abetalipoproteinemia or homozygous hypobetalipoproteinemia and
HDL are the only lipoproteins in their plasma. Apo A-I has also been
proposed as an important structural protein for the biosynthesis of
HDL. Individuals with defects in the apo A-! gene, who thus fail to
biosynthesize apo A-1, have a virtual absence of HDL in plasma and
are at increased risk for developing premature cardiovascular disease.
Apolipoproteins can function as a cofactor or activator of
enzymes involved in lipid-lipoprotein metabolism. Apo C-I1 is required
for the enzymic activity of lipoprotein lipase, the enzyme responsible
for hydrolysis of lipoprotein triglycerides to free fatty acids and
monoglycerides. Patients with a deficiency of apo C- II have severe
hypertriglyceridemia, recurrent bouts of pancreatitis, and eruptive
xanthomas. Apo A-I activates lecithin: cholesterol acyltransferease
(LCAT, EC, phosphatidyl choline-sterol acyltransferase), which
catalyzes the esterification of cholesterol to cholesteryl ester.
Apolipoproteins also play a pivotal role in lipoprotein
metabolism, being the ligand on the lipoprotein particle that interacts
27
with cellular receptors for specific lipoproteins. Apo B-100 and apo E
interact with the LDL receptor to initiate absorptive endocytosis
followed by the catabolism of LDL. Apo E has also been proposed to
interact with the putative remnant receptor, which may play an
important role in removal of hepatic chylomicron remnants by the
liver. Apo A-1 has been proposed to interact with a putative HDL
receptor, and facilitate the removal of cholesterol from peripheral cells
for transport back to the liver. The well-established functions of the
individual apolipoproteins are summarized in Table 2:3 above.
Apolipoproteins play essential roles in maintaining the
structural integrity and functional specificity of plasma lipoproteins.
Because of these properties, apolipoproteins concentrations in plasma
may be used as a new means for characterizing normal and impaired
processes of lipid transport.
The present concept of plasma lipoprotein as a unique
macromolecular system of lipid-protein interactions is based on
pioneering studies of Macheboeuf and his coworkers, who introduced
the concept of constancy of composition and lipid-binding specificity
of protein moieties as the essential chemical requirements for
recognizing and defining individual lipoproteins. As charged
macromolecules, lipoproteins behave on the electric field like typical,
simple proteins. However, because of their lipid components,
lipoproteins have relatively low hydrated densities and behave in the
gravitational field more like lipids than like proteins. Electrical charge
and hydrated density, their most characteristic physical
29
properties,have been utilized as the basis for developing several
electrophoretic and ultracentrifugal procedures for isolating and
Characterizing lipoproteins in plasma, and as operational
criteria for their differentiation and classification. By the late 1950s,
plasma lipoprotein were viewed as macromolecular complexes of non
covalently bound neutral lipids phospholipids, and at least two
apolipoproteins ( - and -proteins) forming discontinuous particle
distributions that were heterogeneous with respect to size, hydrated
density, electric charge, and lipid-protein composition. Whereas
electrophoretic procedures have mainly been used as an analytical
tools for the qualitative and semi-quantitative analysis of lipoproteins
in plasma, sequential ultracentrifugation has become the most
frequently applied procedure for their preparative isolation.
(Alaupovic. et al, (1988). The existence of a metabolic relationship
between major lipoprotein density classes and the clinical usefulness
of electrophoretic and ultracentrifugal lipoprotein patterns for
classifying hyperlipoproteinemias have strengthened the concept that
operationally defined electrophoretic bands and especially, lipoprotein
density classes be accepted and treated as the fundamental physical-
chemical and functional entities for transport of plasma lipids.
Because of their potential significance and involvement in the genesis
and development of atherosclerotic lesions, the lipid constituents of
plasma lipoproteins were studied more thoroughly than the
corresponding protein components. However, during the 1960s, the
discovery of a number of apolipoproteins and characterization of
30
Tangier disease and abetalipoproteinemia as apolipoprotein-deficiency
disorders indicated that apolipoproteins play an essential role in
maintaining the structural integrity and stability of lipoprotein
particles. In addition to their role in the formation of lipoprotein
particles, apolipoproteins perform various functions in the metabolic
conversion of lipoproteins, including their secretion, retardation of
their premature removal, recognition of their binding and removal
sites on cellular surfaces and activation of lipolytic enzymes.
The application of immunological techniques to studies of the
localization and quantification of apolipoproteins has demonstrated a
wide distribution of apolipoproteins throughout the entire lipoprotein
density spectrum. Moreover, the occurrence, within each of the major
lipoprotein density classes, of non equimolar ratios of apolipoproteins
has shown that individual lipoprotein particles of the same density
class may not necessarily have the same apolipoprotein composition.
This seemingly paradoxical relationship has been resolved by
disclosure that operationally defined lipoprotein classes consist of
several discrete lipoprotein particles rather than single, chemically
uniform, lipid-protein complexes.
While revealing another type of heterogeneity in operationally
defined lipoproteins, these findings have also shown that the chemical
uniqueness of apolipoproteins makes them useful as specific markers
for identifying and classifying discrete lipoprotein particles,
irrespective of their density or other nonspecific physical properties.(
Band . et al 1988).
31
Lipoprotein families that contain a single apolipoprotein are
called simple lipoproteins, and those characterized by the presence of
two or more apolipoproteins are referred to as complex lipoproteins.
Simple and complex lipoprotein families can be defined as
polydisperse systems of particles that are heterogeneous with respect
to size, hydrated density, and lipid-protein composition. Such
lipoprotein families are named according to their apolipoprotein
constituents. For example, a lipoprotein family that contains
apolipoprotein (apo) B as the sole protein constituent is called
lipoprotein B (LP-B), whereas a lipoprotein family that contains
apolipoproteins B and E is called lipoproteins B:E (Lp-B:E).The
recognition of apolipoprotein as the essential structural and functional
constituents of lipoprotein particles has created a need for developing
reliable assys for their quantification in plasma and isolated
lipoprotein preparations. Normal functioning of lipid-transport
processes depends on and is reflected in certain optimal concentration
ranges of plasma apolipoproteins or, more precisely, their
corresponding simple and complex lipoprotein families. However,
any perturbations of these processes are expected to result in changed
concentrations of apolipoproteins or lipoprotein families, depending on
the type and extent of underling metabolic defect and (or)
environmental influence. Therefore, each metabolic or environmental
derangement of lipid transport should be characterized by a specific
concentration profile of apolipoproteins.
32
2.8 Factors Affecting Serum Lipid Levels
Natio (1989B) stated that a person’s serum or plasma
cholesterol concentration is under the influence of several factors, viz;
Genetics, Age, Sex, Hormones, physical activity, Diet and Primary
disease states.
Genetics probably have the most important influence on a
person’s cholesterol concentration. Average levels vary substantially
with different ethnic and geographical populations, specific hereditary
traits, number of diseases and a wide range of dietary habits. This fact
is evidenced in the varied reference ranges obtained by researchers
using different racial population (Vartiainem et al, 1982). The genetic
factors were further highlighted by Ehnholm et al (1986) and
Ulermann (1987) who established a relationship between
apolipoprotein E genotypes and cholesterol, which is dependent on
cultural and ethnic background. To this Tikkanen et al (1990) added
that cholesterol elevation during high fat diet is predisposed by
apolipoprotein E4 homozygosity. This effect of apo E genotype on
plasma cholesterol is modulated by dietary fat and cholesterol intake.
Diet also has a very striking effect on the plasma cholesterol
level. Consequently, diet has been used by many workers to adjust
serum cholesterol to a low risk range (Gendy, 1972, Westel, 1979,
NCEP, 1993). There is considerable evidence that the effect on serum
cholesterol level of most of the fats contained in usual human diets
depend mainly on the composition of the fats in terms of saturated
and polyunsaturated fatty acids (Naito, 1989). It is generally accepted
33
that isocalorically exchanged glyceride of saturated fatty acids with
12, 14 and 16 carbon atoms have a cholesterol raising effect whereas
polyunsaturated fatty acid glycerides decrease serum cholesterol level
(Vartiainen, 1982). It has also been shown by Grande (1982) that
isocaloric substitution of glycerides of saturated fatty acids of 12, 14
and 16 carbon atoms for dietary carbohydrates in the extent of 1% of
the total caloric intake causes an average increase in serum
cholesterol of 2.4mg/1ooml. Starvation lowers plasma cholesterol
concentration by lowering hepatic cholesterol synthesis.
Cholesterol concentration in the blood of males always higher
than that in pre-menopausal females Natio (1989b). After menopause,
the cholesterol concentration is higher in females than in males.
Serum cholesterol levels in males seem to reach a plateau by 50 to 60
years of age. Also serum cholesterol concentration starts out around
65mg/100ml at birth and steadily increases with age.
In a study of total, LDL, and HDL cholesterol decrease with age
in older men and women by Bales, (2000) shows that HDL –C levels do
not vary with age. Total Cholesterol LDL – C levels increase with age in
young or middle-aged adults while decrease in adults of 65years and
above. The sex difference in cholesterol concentration reflects the
influence of sex steroids on both body flat distribution and
metabolism (Braunwald, et al, 2001).
Individuals, particularly men who take part in intense physical
exercise have cholesterol values in the lower range (Steinmetz, et al,
1980). This beneficial effect of sports in decreasing cholesterol
34
concentration in plasma seems to be independent of age and
overweight (Bales, 2000), and seems also to include increasing the
efficiency of weight-reducing diets . Thus, physical activity tends to
lower serum total cholesterol. Much of this effect depends on the
type, intensity, duration and frequency of the physical activity.
Exercise, also lowers LDL cholesterol but increases HDL cholesterol
concentrations (Bales, 2000).
Growth hormone, thyroxine and glycagons decrease serum
cholesterol levels whereas anabolic steroids and progestins increase
cholesterol levels (Naito, 1989b). The progestins effect of oral
contraceptive on HDL cholesterol is a particularly controversial
subject, probably because of the qualitative and quantitative
differences between the many formulations of drug combinations in
use (Rossner et al, 1980). However, an experiment carried out by
Gerardo (1980) showed that the cholesterol levels of females taking
sex hormones are higher than those not taking sex hormones
preparations at all ages below the age stratum, 50-54 years. A reversal
occurs at this point, and Females not on hormone have higher
cholesterol levels in the older age groups.
Primary disease states such as diabetes, acute thyroid
dysfunction, obstructive liver disease, acute porphyria nephrotic
syndrome and dysgammaglobulinacmias have an effect on blood
cholesterol concentration. Serum cholesterol levels have been shown
to be increased in hypothyroidism but lowered in hyperthyroidism
(Burtis and Ashwood, 1999) as well as in severe parenchymatous liver
35
damage, infection and anaemia. It has been shown by Aduba et al
(1984) that diabetes mellitus increases plasma cholesterol level. Other
disease conditions as renal failure, gout and hyperuricaemia also
increase plasma cholesterol level. The risk of premature
cardiovascular disease.
2.9 Lipid Metabolism in Diseases
2.9.1 - Lipoprotein (High Density Lipoprotein, HDL). There are no
known diseases characterized by increased levels of -lipoprotein but
it is almost completely absent in a rare inborn error of metabolism,
Tangier disease (named after the island in the U.S.A. where the first
two patients were discovered). Children with this disorder have few
symptoms but cholesterol esters are deposited in the tonsils and
spleen which become enlarged. (Devlin, 1993).
Rarely, low plasma HDL is due to a genetic deficiency of one of
the structural components of HDL (such as Apo A-1). However, low
HDL cholesterol levels are usually the secondary consequence of
increased plasma levels of VLDL and IDL (Intermediate Density
Lipoprotein) (or chylomicrons and their remnants). Mutations in the
ABC I gene are associated with Tangier’s disease, a rare form of low
HDL. Low levels of HDL cholesterol and apo A-I may increase
atherosclerosis risk by any of several mechanisms. HDL could remove
cholesterol from foam cells in atherosclerotic lesions or protect LDL
from oxidative modification. Alternatively, the atherosclerotic risk of
low HDL may be due to the commonly associated elevations of apo B-
36
containing lipoproteins, which accept HDL cholesteryl esters and
deliver cholesteryl esters to the vessel wall (Braunwald et al, 2001).
2.9.2 Tangier Disease: Patients with homozygous tangier disease
have marked HDL deficiency (HDL cholesterol <5mg/dl) associated
with hypercatabolism of HDL constituents, cholesteryl-ester-rich
macrophages (as displayed in enlarged, orange tonsils), intermittent
neuropathy, and premature coronary artery disease (CAD). The
precise defect is not known. Apo A-1 concentrations are about 1% of
the normal value, Apo C-III concentrations are normal. These patients
have mild hypertriglyceridemia and decreased LDL cholesterol.
(Schaefer et al 1988). Isoelectric focusing of plasma proteins
demonstrates increased amounts of pro-apo A-1. Heterozygotes can
also have premature CAD, and their concentrations of HDL cholesterol
and apo A-I in plasma are approximately 50% of normal. The disease
is rare, and is an autosomal co-dominant disorder.
2.9.3 Apolipoprotein A-1 Variants: A number of kindreds have been
reported with moderate HDL deficiency and an abnormal apo A-1
pattern by isoelectric focusing. These variants of apo A-1 include
milano, Marburg, Giesan, and several Munster variants. (Schaefer et
al, 1988). Specific, amino acid substitutions within apo A-1 have been
reported for these variants. The milano variant has not been linked
with premature coronary artery disease.
37
2.9.4 Familial Hypoalphalipoproteinemia: This common autosomal
dominant disorder is characterized by HDL cholesterol concentrations
below the tenth percentile of normal, the disorder has been associated
with a DNA restriction fragment length polymorphism adjacent to the
apo A-1 gene. These patients have normal concentrations of
triglyceride and decreased apo A-1 production. (Schaefer et, al 1988).
Schaefer and colleagues observed this disorder in 4% of patients
with premature coronary artery disease (CAD).
2.9.5 The Beta-lipoproteins (low density lipoproteins, LDL) carry 60
to 70% of the cholesterol in the plasma, high levels of plasma ß-
lipoproteins, and therefore cholesterol, are found in an inherited
disorder, Type II hyperlipoproteinaemia, characterized by premature
arterial disease particularly of the coronary arteries. Increased ß-
lipoprotein levels also result from hypothyroidism and some other
disease low levels of -lipoprotein occur with defects of intestinal
absorption, in starvation and in a rare inherited disorder,
abetalipoproteinaemia, which is caused by defective hepatic synthesis
of ß-apoprotein. A feature of this disorder is a failure of chylomicron
formation in the intestine and therefore defective absorption of fat
(Devlin, 1993).
2.9.6 Familial Hypercholesterolemia(FH) is a codominant genetic
disorder that occurs in the heterozygous form in approximately 1 in
500 individuals. FH is due to mutations in the gene for the LDL
receptor and is genetically heterogenous, >200 different mutations in
38
the gene having been described. Plasma levels of LDL cholesterol are
elevated at birth and remain so throughout life. In untreated adults,
total cholesterol levels range from 1 to 13mmol/l (275 to 500mg/dl).
Plasma triglyceride levels are typically normal, and HDL cholesterol
levels are normal or reduced. As would be expected of a disorder with
decreased numbers of LDL receptors, the fractional clearance of LDL
apo B is reduced. LDL production is increased because the liver
secretes more VLDL and IDL (intermediate density lipoprotein) and
more IDL particles are converted to LDL rather than taken up by the
hepatic LDL receptors. FH heterozygotes usually develop severe
atherosclerosis in early or middle age. Tendon Xanthomas, which are
due to both intracellular and extra cellular deposits of cholesterol,
most commonly involve the Achilles tendons and the extensor tendons
of the knuckles; they are found in about 75% adults with FH.
Tuberous xanthomas, which are softer, painless nodules on the
elbows and buttocks, and xanthelasmas, which are barely elevated
deposits of cholesterol on the eyelids, are common in heterozygous
FH.
The homozygous form of FH occurs in 1 out of 1 million
individuals and is associated with a marked increase of plasma
cholesterol levels (>13mmol/L; >500mg/dl), large xanthelesmas, and
prominent tendon and planar xanthomas. These individuals have
severe, premature CHD (coronary heart disease) that can be
manifested in childhood.
39
2.9.7 Polygenic hypercholesterolemia: Most moderate
hypercholesterolemia (Plasma cholesterol levels between 6.5 and
9mmol/L (240 and 350mg/dl) is polygenic in origin. Multiple genes
interact with environmental factors to contribute to the
hypercholesterolemia, and both over-production and reduced
catabolism of LDL are thought to play roles in the pathophysiology.
The severity is probably affected by the consumption of saturated fat
and cholesterol, age, and the level of physical activity (Braunwald et al
2001) plasma triglyceride and HDL cholesterol levels are usually
normal. These individuals are at increased risk of atherosclerosis.
Tendon xanthomas are not present. Genes involved in cholesterol and
bile acid metabolism may be involved in the pathogenesis (Braunwald
et al, 2001).
2.9.8 Hypertriglyceridemia: The diagnosis of hypertriglyce - ridemia
is made by determining plasma lipids after an overnight fast. Because
of the less certain association of triglycerides with CHD (Coronary
Heart Disease) (Compared to LDL cholesterol), plasma concentrations
greater than the 90th or 95th percentile for age and sex has been used
to define hypertriglyceridemia. Some studies show, however, that
plasma triglyceride levels >130 to 150mg/dl are associated with low
HDL cholesterol levels and small, dense LDL particles.
Elevations in plasma triglycerides are usually associated with
increased synthesis and secretion of VLDL triglycerides by the liver.
Hepatic triglyceride synthesis is regulated by substrate flow (the
40
availability of free fatty acids), energy balance (the level of glycogen
stores in the liver), and hormonal status (the balance between insulin
and glucagons) (Burtis and Ashwood, 1999) Obesity, excessive
consumption of simple sugars and saturated fats, inactivity, alcohol
consumption, and insulin resistance are commonly associated with
hypertriglyceridemia (Braunwald et al 2001). In most of these
situations; increased free fatty acid flux from adipose tissue to the
liver stimulates the assembly and secretion of VLDL. The addition of
chylomicrons to the circulation may cause dramatic increases in
plasma triglycerides. Isolated elevations of plasma triglycerides can be
due to increased levels of VLDL (type IV) or combinations of VLDL and
chylomicrons (type V). Rarely, only chylomicron levels are elevated
(type 1) (Braunwald et al, 2001) plasma is usually clear when
triglyceride levels are <4.5mmol/L (<400mg/dl) and cloudy when
levels are higher and VLDL (and /or chylomicron) particles become
large enough to scatter light. Pancreatitis is the major risk associated
with plasma triglyceride concentrations >11mmol/L (>1000mg/dl)
when VLDL triglyceride levels are markedly elevated (>11.5mmol/L
(>1000mg/dl) Lipoprotein Lipase) may be saturated so that an
acquired LPL deficiency develops during the postprandial period even
if there is no underlying genetic disorder.
2.9.8.1 Pre-B-lipoproteins: are mainly formed in liver and, to a
smaller extent, by the mucosal cells of the small intestine, their main
role is in the transport of endogenous triglyceride from the liver to the
41
sites of its utilization for energy production (as in muscles) or storage
(as in adipose tissue). Plasma levels of pre-B-lipoproteins are
increased in an inherited disorder hyperlipoproteinaemia type IV and
in a number of diseases including diabetes mellitus and
hypothyroidism and also after an excessive intake of alcohol. (Devlin,
1993).
2.9.8.2 Familial hypertriglyceridemia: This appears to be
transmitted as an autosomal dominant disorder, though the
underlying mutation(s) have not been identified. The pathophysiology
is complex: both reduced catabolism of triglyceride-rich lipoproteins
and over production of VLDL have been reported. Elevated levels of
fasting plasma triglycerides in the range of 2.3 to 8.5mmol/L (200 to
750mg/dl) are usually associated with increased levels of VLDL
triglycerides only.
2.9.9 Secondary causes of Hyperlipoproteinemia
2.9.9.1 Diabetes Mellitus: Diabetes can affect lipid and lipoprotein
metabolism through several mechanisms. In Type 1 diabetes mellitus
(DM) (Formerly called insulin-dependent diabetes mellitus), plasma
lipids are usually normal when control of diabetes with insulin is
adequate. In diabetic ketoacidosis, hypertriglyceridemia can be severe
due to increases in both VLDL and chylomicrons. These
abnormalities are associated with overproduction of VLDL and LPL
42
deficiency secondary to insulinopenia. They usually improve with
tight control of the diabetes. In Type 2 DM (formerly called non-
insulin-dependent diabetes mellitus),insulin resistance and obesity
combine to cause mild to moderate hypertriglyceridemia and low HDL
cholesterol levels. In general, this pattern of dyslipidemia is due to
overproduction of VLDL. LDL cholesterol is usually normal in Type
2DM, though the LDLs are small, dense, and perhaps more
atherogenic. It is recommended that patients with diabetes should be
treated as if they already have CHD, i.e. the treatment goal is to
reduce their LDL to <2.6mmol/L (<100mg/dl).
2.9.9.2 Hypothyroidism: This accounts for about 2% of all cases of
hyperlipidemia and is second only to Diabetes Mellitus as a cause of
secondary hyperlipidemia. Levels of LDL cholesterol can be elevated,
even in patients with subclinical disease in whom thyroid-stimulating
hormone (TSH) levels are elevated but other thyroid function tests are
normal. Hypertriglyceridemia can occur if obesity is present.
Hypothyroidism is also associated with increased levels of HDL
cholesterol, probably because of reduced Hepatic Triglyceride Lipase
activity. Correction of hypothyroidism reverses the lipid abnormalities.
2.9.9.3 Renal Disease: Renal disease causes a wide range of lipid
abnormalities. The nephritic syndrome can be accompanied by
elevations in LDL, VLDL, or both. The severity of the hyperlipidemia
correlates with the degree of hypoproteinemia. Renal failure is
43
associated with hypertriglyceridemia and low HDL cholesterol
concentrations.
2.9.9.4 Ethanol: The metabolism of ethanol enhances the level of
NADH in the liver which, in turn, stimulates the synthesis of fatty
acids and their incorporation into triglycerides. Moderate ethanol
consumption raises plasma VLDL levels, with the degree of elevation
dependent on the baseline level. Severe hypertriglyceridemia and
pancreatitis usually develop on the background of a genetic
hyperlipidemia and heavy alcohol intake. Because ethanol also
stimulates the synthesis of apo A1 and inhibits CETP(cholesteryl ester
transfer protein), ethanol-associated hypertriglyceridemia is usually
accompanied by normal or elevated levels of HDL cholesterol.
2.9.9.5 Liver Disease: Primary biliary cirrhosis and extrahepatic
biliary obstruction can cause hypercholesterolemia and elevated levels
of plasma phospholipids associated with increased levels of an
abnormal lipoprotein and LDL. Severe liver injury often leads to a
decrease in levels of both cholesterol and triglyceride (Braunwald et al
2001).
2.10 Lipid Metabolism in Enteric Fever
Lipid Profile in Enteric Fever: Lipid profile is known to alter in
patients with severe sepsis (Khosla et al 1991). Few studies regarding
the status of lipid levels in enteric fever are available. According to the
study conducted by (Khosla et al) in 1991 about Twenty patients with
44
enteric fever, belonging to different age groups and both sexes, along
with an equal number of matched patients with fever due to non-
enteric causes, were studies with regard to alterations in lipid profile.
They observed a severe and protracted hypertriglyceridaemia, decrease
in HDL-cholesterol levels and increase in LDL-Cholesterol levels in
patients with enteric fever at the peak of fever. The values returned to
normal on recovery and convalescence.
2.11 Normal Expected Lipid Values
Plasma lipid values depend on many factors, notably age and
sex (Baron, 1988; Naito, 1989B) other factors to consider are diet
(Tilkian et al, 1979) the population
sampled, and the specificity of the analytical method used (Baron,
1988).
Using an enzymatic procedure, (Tilkian et al 1979) and (Baron,
1988) established an overall reference range for adults as 4.0-
6.5mmol/L, varying with the population samples and increasing with
advancing age until age 50 years, and being higher in males.
At birth, plasma cholesterol concentration is about 66mg/dl
(1.7mmol/L) and equally distributed among LDL and HDL, with a very
small amount in VLDL. Triglycerides (TG) concentration is about
36mg/dl (0.41mmol/L).
Lipid, lipoprotein and apolipoprotein concentration rise sharply
during the first few months of life. With LDL becoming the major
carrier of plasma cholesterol, and then remaining relatively
45
unchanged until puberty. A profile consisting of total cholesterol of
about 155mg/dl (4.01mmol/L), LDL cholesterol (LDL-C) of 90mg/dl
(2.33mmol/l), HDL cholesterol (HDL-C) of 53mg/dl (1.38mmol/L), TG
of 55mg/dl (0.62mmol/L), apo B-100 of 86mg/dl and apo A-1 of about
130mg/dl is typical for pre pubertal individual (Burtis et al, 1999).
After puberty, an increase in TG, LDL-C and Apo B-100 occurs in both
sexes and a decrease in HDL-C and apo A-I occurs in men. Lipid
concentrations continue to increase throughout adult life, with total
and LDL-C and apo B-100 being higher in men than women up to
55years of age. Thereafter, women who are not receiving estrogen
supplementation have higher total and LDL-C and apo B-100 than
their age-matched male counterparts (National Cholesterol Education
Program (NCEP, 1988).
Cooper, (1982) in a study of the distribution of total cholesterol
values established sex and age specific reference ranges for total
serum cholesterol, for cord blood, it is 1.0-2.5mmol/L irrespective of
sex. The range then increases steadily from 2.6-4.9mmol/L at 0-1year
of age to 4.2-7.4mmol/L at 50-54years for males. For females, the
values are 2.9-5.2, and 4.3-7.6mmol/L for the respective age range.
For females, the values also increases to a 4.6-8.0mmol/L at age 55-
59years. However, there is a non-significant decrease in values for
males of the same age group. Using Nigerian subjects, (Aduba et al,
1984) established serum total cholesterol reference range of 2.9 –
4.9mmol/L for males, and 3.8-5.8mmol/L for females.
46
The combined reference range for fasting plasma triglycerides is
put by Baron, (1988) as 0.3-1.8mmol/L, this being a combination of
exogenous and endogenous triglycerides in transport Wooton and
Freeman (1982) gave a range of 0.7-2.1mmol/L for males and 0.6-
1.5mmol/L for females using young adult Caucasian population.
(Aduba et al 1984) established a reference range for HDL-
cholesterol of 0.6-1.2mmol/L, and 0.9-1.5mmol/L for healthy adult
males and females respectively, while values above 0.9mmol/L and
below 3.4mmol/L and 5.2mmol/L for HDL, LDL and total cholesterol
respectively were reported to be desirable for health [Expert panel
Report (1993)].
The National Cholesterol Education Program has defined a
plasma triglyceride >250mg/dl and a low-density lipoprotein (LDL)
cholesterol value >160mg/dl as increased, and a high density
lipoprotein (HDL) cholesterol concentration <35mg/dl as decreased.
Increased concentration of LDL cholesterol and decreased
concentrations of HDL cholesterol have been associated with an
increased risk for premature coronary artery disease. [Burtis and
Ashwood, 1999].
The significant age-related increases in plasma LDL cholesterol
and apo B values are probably due to age related decreases in LDL
receptor activity and to the high U.S. dietary intake of cholesterol and
saturated fat. Females have significantly (P<0.001) higher
concentrations of HDL cholesterol and apo A-I than do males, in part
because of increased estrogen-mediated production of apo A-I. In
47
addition, females have significantly (P<0.001) lower apo B values than
do males. Apo B values increase significantly (P<0.01) in
postmenopausal women. Menstrual cycle phase can also affect lipids,
with triglycerides values being significantly (P<0.01) higher during the
ovulatory phase than at other times. An apo B value one standard
deviation (ISD) above the mean for middle-aged men is approximately
120mg/dl, while for women this value is about 105mg/dl. An apo A-1
value ISD below the mean is approximately 105 and 110mg/dl for
men and women, respectively. These data are based on the Framing-
ham offspring study (n=3800), for which results were obtained with
sensitive enzyme-linked immunoassays standardized with reference
material from the centers for disease control. (Schaefer et al, 1988).
The advantage of apolipoprotein, in addition to the precision with
which they can be determined, is the fact that their concentrations in
plasma change very little between fasting and non-fasting states.
48
CHAPTER THREE
MATERIALS AND METHODS
3.1 Subjects
Subjects were recruited from University of Nigeria Teaching
Hospital Ituku-Ozalla(UNTH), Parklane Specialist Hospital Enugu,
Ntasiobi-ndi-no-na-afufu Hospital and Annuciation Hospital Enugu.
A total of three hundred (300) adults between the age groups of 20-
40years were involved in the study. Two hundred (200) individuals of
which were eighty(80) males and one hundred and twenty(120)
females were positively diagnosed of enteric fever while one
hundred(100) (50 males and 50 females) were healthy individuals
used as controls.
These subjects were first given the structured questionnaire to
fill. The questionnaire contains the information about their age, sex,
health condition (that is whether they are diabetic, hypertensive or
having renal problems) and whether they have been on any
medication (antibiotic) within the time of the enteric fever.
3.2 Sample Collection: Fasting blood samples were collected from
the chosen subjects by veine-puncture from the median cubital vein
into a clean-labeled plain tube. Subjects that have malaria, Diabetes
Mellitus, Hypertension, Obesity and Renal problems were excluded.
Exclusion was based on information in the questionnaire they filled,
and by running their MP test, checking their Blood pressure, Blood
sugar(using Accu-chek Active-a diabetes monitoring kit) and
49
BMI(Body Mass Index). The fasting blood samples were immediately
tested for typhoid fever by performing the slide and tube agglutination
tests using Cromatest-febrile antigen kit. Also Enterocheck WB kit
from Zephyr Biomedicals India were used as a confirmatory test. The
rest of the blood were allowed to stand for about one to two hours
then centrifuged for 5mins and the sera were separated from the cells
prior to analysis. When immediate analysis was not possible, the
separated sera were deep frozen at – 200c and analysed within five (5)
days. The sera were analyzed for lipid profiles using lipid profile kit
(Biosystems Reagents & Instruments from costa Brava,30 Barcelona
Spain).
3.3 Procedure for analysis:
3.3.1 Slide agglutination test
1. 8 drops of serum was put into the glass slab in 2 rows and 2
columns
2. The commercially prepared standardized antigens of
salmonella typhi and salmonella paratyphi A,B,C of both O
(somatic) and H (flagella) Antigens (febrile Antigen Kit) were
used.
3. A drop of the above reagent (febrile Antigen Kit) for both 0
and H antigens of salmonella typhi and S.paratypli ABC was
dropped near each of the serum in the glass slab.
50
4. The contents of each circle (the serum and the reagent were
mixed together using a disposable separate stirrer for each
circle.
5. The glass slab was rocked gently by hand for 2mins.
6. The glass slab was then observed for any degree of
agglutination.
3.3.2 Tube agglutination test
1. 8 test-tubes were placed in a rack.
2. 4 volume of saline was added to tube 1 and 1 volume to tubes
2-8.
3. I volume of serum was added to tube I and mixed making the
dilution 1 in 5.
4. I volume of serum/saline solution was transferred to tube 2
(Giving 1 in 10 dilution).
4. The procedure in 4 was continued to tubes 3, 4 up to tube 8
making a serum doubling dilutions of 1 in 5, 1 in 10, 1 in 20, 1
in 40, 1 in 80, 1 in 160, 1 in 320 and 1 in 640 respectively.
5. A fresh pipette was used to transfer 0.5ml (starting from highest
dilution) from each test-tube into a corresponding agglutination
tube rack.
6. 0.5ml of antigen was added to each tube.
7. To another agglutination tube 0.5ml of saline and 0.5ml of
antigen were added, this tube serves as a control.
51
8. The agglutination rack was placed in the water bath and the
water level was adjusted until it covers one-third of the tube.
9. Somatic (0) antigens was incubated at 48-50oc for 4 hours while
flagellar (H) antigens was incubated at 48-500c for 2 hours.
Thereafter the agglutination rack was brought out and
examined macroscopically for agglutination.
10. Then those sample with the high titre like typhi 0 1/160 and
above and typhi H 1/160 were used for lipid profile test.
3.4 Enterocheck-wb was also used as a confirmatory test.
3.4.1 Principle of Enterocheck-wb
It utilizes the principles of immunochromatography, a unique
two site immunoassay on a nitrocellulose membrane. The conjugate
pad contains two components – Anti human IgM antibody conjugated
to colloidal gold and rabbit IgG conjugated to colloidal gold. As the
test specimen flows through the membrane test assembly, the highly
specific anti human IgM antibody-colloidal gold conjugate complexes
with the S. typhi specific IgM antibodies in the specimen and travels
on the membrane due to capillary action along with the rabbit IgG-
colloidal gold conjugate. This complex moves further on the
membrane to the test region (T) where it is immobilized by the S. typhi
specific lypopolysaccharide antigen coated on the membrane leading
to formation of a pink to pink-purple coloured band. The absence of
this coloured band in the test region indicates a negative test result.
The unreacted conjugate and unbound complex, if any, move further
52
on the membrane and are subsequently immobilized by the anti-
rabbit antibodies coated on the membrane at the control region (C),
forming a pink to pink-purple coloured band. This control band acts
as a procedural control and serves to validate the results.
3.4.2 Procedure
1. Enterocheck-wb uses human serum/plasma/whole blood as
specimen.
2. The kit components of enterocheck-WB device were brought to
room temperature.
3. The foil pouch was opened by tearing along the “notch”.
4. The testing device and the sample loop were brought out for
immediate use.
5. The device was labeled with specimen identity.
6. The testing device was placed on a flat horizontal surface.
7. 5µl of serum was carefully dispensed into the specimen port
“A”, using a micropipette or the sample loop provided. A dip
was made into the sample container and blot the sample in
the sample port “A”.
8. Five drops of sample running buffer was added into the
reagent port “B”.
9. The result was read at the end of 15 minutes.
10. Negative result (that is if IgM antibodies to S. typhi are not
present) only one coloured band will appear in the control
window (c).
53
11. Positive result (that is if IgM antibodies to S.typhi are
present) two coloured bands will appear in the Test (T and
control windows (C).
3.5 Lipid profile estimation
3.5.1 ESTIMATION OF SERUM CHOLESTEROL
Serum cholesterol was assayed using enzymatic colorimetric
method of Allain, et al(1974).
3.5.1.1 PRINCIPLE OF THE METHOD
Cholesterol esters in the sample are hydrolysed ezymatically to
cholesterol and fatty acids. In the presence of oxygen, the cholesterol
produced and the free cholesterol in the sample are oxidized by
cholesterol oxidase to cholestenone and hydrogenperoxide. The
hydrogenperoxide formed is detected by a chromogenic oxygen
acceptor(phenol-ampyrone,PAP), in the presence of peroxidase and
phenol. The red quinoneimine formed is proportional to the amount of
cholesterol present in the sample.
Cholesterol ester + H2 0 lipase cholesterol + fatty acid
cholesterol + 1/202 + H20 Chol. Oxidase Cholestenone+H20
2H202 + 4-Aminoantipyrine + Phenol Peroxidase Quinone imine +
4H20.
54
3.5.1.2 Procedure
The reagent was brought to room temperature.0.02ml(20µl) of water,
serum and cholesterol standard solution(5.18mmol/l) were added to
clean tubes labeled blank, test and standard respectively. To each
tube 2.0ml of the working reagent was added. The tubes were mixed
well and incubated at room temperature for 10minutes. The
absorbance A of the test and standard were measured at 520nm after
adjusting the instrument (Jenway Spectrophotometer 6100) with the
blank. The controls were treated exactly the same way as the test and
standard.
Calculation
Absorbance of Test x 5.18mmol/L = mmol/L of cholesterol
Absorbance of standard 1
3.5.2 Triglyceride Method
The method employed was the method of Fossati and Prencipe
(1982). The method involves the enzymatic hydrolysis and
quantification of triglyceride which is specific and not subject to
interference by phospholipids.
3.5.2.1 Principle:
Triglyceride is hydrolysed by lipase to form fatty acids and glycerol.
The glycerol concentration is then determined by enzymatic assay.
Coupled with Trinder’s reaction that terminates in the formation of a
55
quinoneimine dye. The amount of the dye formed, determined by its
absorption at 520nm is directly proportional to the concentration of
triglycerides in the sample.
Triglycerides + H20 Lipase Glycerol + Fatty acids
Glycerol + ATP Glycerol Kinase Glycerol -3-P+ADP
Glycerol -3-P+02 G-3-P-oxidase Dihidroxyacetone - P +H202
2H202+4-Aminoantipyrine + 4 – Chlorophenol Peroxidase
Quinoneimine + 4H20
3.5.2.2 Procedures
1.0ml of freshly reconstituted reagents (Biosystem reagents) was
added into tubes labeled test, standard and blank and incubated at
370c water bath for 5minutes. 0.01(10µl) of sample, standard and
water were added to respective tubes, mixed and incubated for
5minutes at 370c. The absorbance (A) were read
spectrophotometrically at 520nm, using blank to zero the instrument.
The final colour is stable for at least 2hours at room temperature.
Controls were treated as test.
3.5.2.3 Calculation
Absorbance of Test x 2.26
Absorbance of standard 1 = mmol/l triglyceride
56
3.5.3 Estimation of serum high density lipoprotein (HDL)
cholesterol:
The phosphotungstate/magnesium chloride oxidase/peroxidase
method for the determination of HDL cholesterol as suggested by
(Burnstein et al (1970) was used.
3.5.3.1 Principle:
LDL and VLDL are precipitated from serum by the action of a
polysaccharide(Heparin) in the presence of divalent cations. Then,
HDL-C present in the supernatant is determined using the method of
Fredrickson et al, (1967).
3.5.3.2 Procedure
To 0.2ml of sample in a clean centrifuge tube was added 0.5ml of
reagent A. The tube was mixed and allowed to stand for 30minutes at
room temperature and then centrifuged for 10minutes at a minimum
of 4000 r.p.m. The supernatant (50µl) was carefully transferred into
another tube and cholesterol was then determined using the method
described above.
3.5.3.3 Calculation
Absorbance of Test x concentration of standard x Dilution factor
sample Absorbance of standard 1
= A of Test x 1.36mmol/L = HDL Cholesterol
A of Standard 1
57
3.5.4 Estimation of very low density lipoprotein (VLDL)-
cholesterol
VLDL was estimated using Friedewald formular (Friedewald et al 1977)
VLDL in mmol/L = Triglyceride 2.2
3.5.5 Estimation of low density lipoprotein(LDL)-
cholesterol
LDL was estimated using Friedewald formular (Ogedegbe, 1996) LDL
mmol/L = Total cholesterol – HDL – VLDL.
58
CHAPTER FOUR
RESULTS
The mean values (±SD) of lipid profile for patients and controls were
TG = 1.4 + 0.47, 1.32+ 0.31, TC = 4.54 + 1.15, 4.49 + 0.95, HDL – C =
1.19+ 0.39, 1.24+ 0.31, LDL –C = 2.73 + 1.03, 2.65 + 0.82, VLDL –C =
0.64 + 0.21, 0.60 + 0.14 respectively. There were no significant
differences (p > 0.05) between these values. (see table 4.1)
The mean values (±SD) of male patients and male control TG =
1.37+ 0.42, 1.27+ 0.31,
TC = 4.74+ 1.56, 4.22+ 0.92,
HDL –C= 1.25+ 0.42, 1.23 + 0.34,
LDL – C= 2.87+ 1.34, 2.42 + 0.71,
VLDL-C = 0.62+ 27.49, 0.57+ 0.14.
This shows statistically significant differences (p < 0.05) in TC and
LDL–C (i e there was significant increase in the TC and LDL-C of the
male patients against the male control) while there were no
significant difference in the other parameters (TG, HDL-C, VLDL–C)
(see table 4.2) . However, there were also significant difference
(p<0.05) in total cholesterol and LDL–C of female patients and controls
(i e female control increases against patients).
TC = 4.41 + 0.75, 4.77 + 0.89,
LDL–C= 2.62 + 0.74, 2.90 + 0.86 (see table 4.3).
The relationship between the lipid profile and age of patients
and controls were compared.
59
Fig 1-10) The following parameters correlated positively with age
Triglycerides of patients and age (r=0.67, P <0.001) (fig. 1). Serum
VLDL–cholesterol of patients and age (r = 0.66, P < 0.0001) ( fig 5),
Triglycerides and age of controls (r = 0.79, P <0.001) fig 6), VLDL–C of
control and age ( r = 0.79, P < 0.001) ( fig 10) and negatively HDL–C
and age of patients ( r = -0.27, P < 0.0001) ( fig 3), LDL and age of
controls ( r= -0.18, P<0.117) ( fig 4), TC and age of controls ( r = -0.37,
P <0.0002) (fig 7), HDL–C and of controls (r = -0.45, P< 0.001) (fig 8),
and LDL-C of control and age (r = -0.39, P<0.0001) (fig 9). While there
was no significant correlation between total cholesterol of patients and
age (r= -0.13, p = 0.0685) (fig 2).
Furthermore, the titre of somatic (0) antigen were compared
with lipid profile of patients and controls (figs 11-20). The following
parameters correlated with titre of somatic (0) antigen Triglyceride of
patients and titre ) (r = 0.25, P <0.0003) (fig 11), VLDL–C and titre 0
of patients ( r = 0.21, P = 0.0025) fig 15),
LDL-C and the titre 0 of patients ( r = -0.16, P = 0.0213) ( fig 14).
While there were no significant correlation between TC and titre of
patients ( r= -0.09, P = 0.1831) (fig12) and HDL- C of patients and titre
0 (r=0.02, p = 0.804) (fig 13).
The age of the patients and controls were grouped in fives and
were compared with all the parameters (fig 21 and 22). This histogram
shows that serum triglyceride and VLDL were increasing with age
while LDL–C and HDL–C were decreasing with age, and no difference
in total cholesterol (fig 21). However, there was difference in total
60
cholesterol of control and age which was decreasing with age as well
as LDL-C and HDL–C (fig 22) while Triglyceride and VLDL–C were
increasing with age (fig 22).
61
Table 4.1
Lipid profile of enteric fever patients and of controls
TG(mmol/L)
TC
(mmol/L)
HDL-C
(mmol/L)
LDL-C
(mmol/L)
VLDL-C
(mmol/L)
Patients
n=200
1.4 + 0.47
4.54 +
1.15
1.19+
0.39
2.73+1.03
0.64+0.21
Controls
n=100
1.32+0.31
4.49+0.95
1.24+0.31
2.65+0.82
0.60+0.4
P-values 0.0904 0.6694 0.2168 0.5365 0.056
Significant NS NS NS NS NS
Mean ± SD
NS = Not Significant (when P>0.05)
SG = Significant (when P<0.05)
62
Table 4.2
Lipid profile levels of male patients and age-matched controls
TG(mmol/L)
TC (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
VLDL-C (mmol/L)
Patients
n=80
1.37+0.42
4.74+1.56
1.25+0.42
2.87+1.34
0.62+27.49
Controls
n=50
1.27+0.31
4.22+0.92
1.22+0.34
2.42+0.71
0.57+0.14
P-values 0.1598 0.0343 0.7878 0.029 0.159
Significant NS SG NS SG NS
Mean ± SD
63
Table 4.3
Lipid profile of female patients and age – matched controls
TG(mmol/L)
TC (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
VLDL-C (mmol/L)
Patients
n=120
1.42+0.49
4.41+0.75
1.16+0.36
2.62+0.74
0.65+0.22
Controls
n=50
1.38+0.31
4.77+0.89
1.26+0.26
2.90+0.86
0.62+0.14
P-values 0.5655 0.009 0.068 0.0381 0.4221
Significant NS SG NS SG NS
Mean ± SD
64
Table 4.4
Lipid profile of patients (m & f) and age-matched controls(male & female) grouped according to age (in fives).
Means of parameters (mmol/L)
Age
groups
Patients (mmol/L
Controls (mmol/L)
TG TC HDL-C LDL-C VLDL-C TG TC HDL-C LDL-C VLDL-C
20-25 1.12 4.67 1.30 2.86 0.51 1.15 4.76 1.33 2.91 0.52
26-30 1.51 4.36 1.07 2.63 0.66 1.50 4.20 1.19 2.35 0.67
31-35 1.81 4.48 1.14 2.52 0.83 1.87 3.93 1.0 2.09 0.85
36-40 1.84 4.41 1.06 2.53 0.83 1.86 3.63 0.87 1.91 0.85
P = 0.9995
F-ratio = 0.005032 (Not Significant)
65
TABLE 4.5
Correlation of parameters ( TG, TC, HDL-C, LDL-C, VLDL-C)
With age and Titres O and H for Patients
AGE TITRE O TITRE H
Parametres (r)
Pearson
P-values Sig. (r )
Pearson
P-
Values
SIG (r )
Pearson
P-values SIG.
TG 0.67 0.0001 SIG 0.25 0.003 SIG 0.09 0.1949 NS
TC -0.13 0.0685 NS -0.09 0.1831 NS 0.01 0.8752 NS
HDL-C -0.27 0.0001 SIG 0.018 0.804 NS -0.03 0.6581 NS
LDL-C -0.18 0.0117 SIG -0.16 0.0213 SIG -0.005 0.9353 NS
VLDL-C 0.67 0.0001 SIG 0.21 0.0025 SIG 0.07 0.321 NS
66
CHAPTER FIVE
Discussion
The result presented showed no significant difference in all the
parameters of the lipid profile for patients and controls (male and
female). This does not agree with the work of Khosla et al (1991)
which reported elevated levels in LDL – cholesterol and triglycerides
of patients with acute enteric fever. This difference may be attributed
to the fact that different population of patients with different
environmental factors at play were involved in the two studies.
However, there exist sex-related difference in the total
cholesterol and LDL – C of the male and female subjects. In the males
there was a significant difference showing an increase in the total
cholesterol (TC) and low-density lipoprotein cholesterol (LDL – C) of
the patients against the control. While there was an opposite case in
the female category which shows a significant increase in the total
cholesterol and LDL-C of the controls against the patients. This high
levels of total cholesterol and LDL – cholesterol in male patients may
be as a result of decreased concentration of LDL – receptors in the
liver ( Burtis et al, 2001). Since LDL –receptors help in the removal of
LDL – C from circulation or it could be as a result of reduced LDL
binding because of defective/absence of LDL – receptors.
This study also shows an increase in the total cholesterol and
LDL – C of male patients against female patients. This agrees with the
report (National cholesterol Education Program, 1998) that before
menopause, women tend to have total cholesterol levels lower than
men at the same age. (Considering the age groups used in this study).
While menopause is often associated with increase in LDL cholesterol
in women. It can also be said that total cholesterol and LDL –
67
cholesterol is sex –influenced. Again, as total cholesterol is increasing
LDL–C is also increasing. This may be as a result of the constituents
of LDL–C which is mainly cholesterol and little protein (Burtis et al,
1999). Again, it seems as if the male patients in this study do not take
part in intense physical exercise as Steinmetz, et al, 1980 showed in
their study that individuals, particularly men who take part in intense
physical exercise have cholesterol values in the lower range.
The relationship between the lipid profile of patients and age
were compared. There exist a relationship between Triglyceride and
VLDL–cholesterol. This is shown in this study as there exist a
significant positive correlation between serum triglycerides of patients
and age, VLDL–C of patients and age, Triglycerides of controls and
age, VLDL–C of controls and age. This may be as a result of the
constituents of VLDL–C which is mainly triglyceride (Burtis et al,
1999). However, total cholesterol of control and age showed significant
negative correlation that is total cholesterol was decreasing with age.
This is not in line with much literature on total cholesterol levels and
age (Burtis et al, 1999, National Cholesterol Education Program,
1998) that state increase in total cholesterol within middle ages of 20-
50years and decreases from 65years above. The serum triglyceride
and VLDL- C decrease with age this suggests that all the parameters
Triglyceride, Total Cholesterol, HDL –Cholesterol, LDL–C and VLDL–C
are age –influenced.
68
Conclusion
Studies in lipid and lipoprotein abnormalities are common in
many diseases like diabetes, alcoholism, renal problems,
atherosclerosis and others but not in enteric fever. Very few studies
regarding the status of lipid levels in enteric fever are available.
In this study it was shown that there is no alteration in the lipid
profile of enteric fever patients. Total cholesterol and LDL-C has been
shown to be both gender and age influenced. However, it is suggested
that more research work be conducted in this area using other
confirmatory tests for typhoid fever identification other than the ones
used in this study like stool culture, blood culture and bone marrow
culture to compare the result. Also, the incidence of typhoid fever can
be reduced by the improvement in hygience of disposal of human
wastes, water supplies and food preparation. Katung (2000) state that
the most cost-effective strategy for the control of typhoid fever in the
developing countries is to implement public health measures to
provide clean water supplies and sanitary disposal of excrete.
69
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75
APPENDIX 1
RAW DATA
MALE AND FEMALE SAMPLE RESULT OF PATIENTS WITH TITRE
ABOVE 160.
S/NO TG TC HDL LDL VLDL SEX AGE(yrs)
1 1.45 4.93 0.98 3.29 0.66 m 21
2 1.21 4.93 1.17 3.21 0.55 m 20
3 1.21 3.56 1.05 1.96 0.55 f 20
4 1.13 4.52 0.86 3.15 0.51 f 21
5 1.45 3.83 1.30 1.87 0.66 f 39
6 1.37 3.83 0.62 2.59 0.62 m 40
7 1.37 3.01 0.56 1.83 0.62 m 38
8 1.13 3.28 0.86 1.91 0.51 m 40
9 1.04 4.38 1.42 2.49 0.47 f 21
10 1.37 4.65 1.48 2.55 0.62 f 38
11 1.37 4.11 1.05 2.44 0.62 m 40
12 1.08 3.28 1.02 1.77 0.49 f 20
13 1.45 3.56 1.17 1.73 0.66 f 39
14 1.13 3.83 0.80 2.52 0.51 f 40
15 1.33 5.2 1.55 3.05 0.60 f 37
16 1.93 3.28 0.49 1.91 0.88 f 38
17 1.53 4.11 1.24 2.17 0.70 f 40
18 1.69 3.83 0.93 2.13 0.77 m 40
19 1.13 4.65 1.42 2.72 0.51 f 22
20 1.29 3.28 1.48 1.21 0.59 m 39
76
21 1.45 4.11 1.67 1.78 0.66 f 38
22 1.29 5.2 1.48 3.13 0.59 f 40
23 1.21 4.93 0.98 3.4 0.55 f 21
24 1.53 4.65 0.62 3.33 0.70 f 36
25 1.45 3.83 0.98 2.19 0.66 m 35
26 3.3 3.3 0.4 1.4 1.5 f 40
27 0.9 6.1 1.6 4.1 0.4 m 20
28 1.0 4.3 1.9 1.9 0.5 f 21
29 1.1 4.1 1.4 2.2 0.5 f 22
30 1.1 4.1 1.6 2.0 0.5 f 23
31 1.5 5.2 1.3 3.2 0.7 m 37
32 1.1 4.6 1.7 2.4 0.5 f 23
33 3.1 2.8 0.5 0.9 1.4 f 39
34 2.3 3.3 0.5 1.7 1.1 f 36
35 1.5 5.6 1.3 3.6 0.7 m 35
36 0.9 6.6 2.5 3.7 0.4 m 20
37 1.6 4.8 0.7 3.4 0.7 f 30
38 1.2 5.0 1.6 2.8 0.6 f 21
39 1.0 4.6 1.3 2.8 0.5 f 22
40 3.1 11.7 1.4 8.9 1.4 m 36
41 1.1 4.8 0.9 3.4 0.5 f 21
42 1.2 4.8 1.1 3.2 0.5 f 24
43 1.0 5.6 1.5 3.6 0.5 m 20
44 1.5 5.4 1.5 3.2 0.7 m 36
45 1.5 5.0 1.5 2.8 0.7 f 35
77
46 1.1 4.4 1.2 2.7 0.5 f 20
47 1.4 3.5 0.6 2.3 0.6 f 26
48 1.0 5.0 1.4 3.1 0.5 m 21
49 1.1 5.0 1.4 3.1 0.5 m 22
50 1.8 4.8 1.5 2.5 0.8 f 34
51 1.0 6.2 1.7 4.0 0.5 m 22
52 1.6 3.7 0.8 2.2 0.7 f 36
53 1.1 4.8 1.1 3.2 0.5 m 23
54 1.0 4.2 1.0 2.7 0.5 f 22
55 1.4 3.6 1.4 1.6 0.6 m 35
56 1.1 2.8 0.7 1.6 0.5 f 22
57 1.6 6.8 0.9 5.2 0.7 m 36
58 1.6 3.2 0.7 1.8 0.7 m 37
59 1.6 4.2 1.5 2.0 0.7 m 39
60 1.3 5.2 1.8 2.8 0.6 m 25
61 1.2 5.0 1.5 3.0 0.5 f 22
62 1.1 5.4 1.2 3.7 0.5 m 22
63 2.4 4.2 2.0 1.1 1.1 f 40
64 1.2 5.2 2.2 2.5 0.5 m 24
65 1.2 3.6 1.1 2.0 0.5 f 22
66 1.8 4.2 1.0 2.4 0.8 f 30
67 1.2 5.2 1.6 2.7 0.5 m 21
68 1.1 4.6 1.7 2.4 0.5 m 21
69 2.2 5.6 1.4 3.2 1.0 f 39
70 2.4 5.4 1.1 3.2 1.1 f 37
78
71 1.2 3.2 1.2 1.5 0.5 m 21
72 1.3 4.6 0.9 3.1 0.6 f 25
73 1.4 3.4 0.6 2.2 0.6 f 29
74 1.6 3.8 1.2 1.9 0.7 f 30
75 1.3 5.2 1.4 3.2 0.6 m 24
76 1.3 4.6 1.3 2.7 0.6 m 26
77 1.1 5.0 1.3 3.2 0.5 f 21
78 2.0 3.0 0.8 1.3 0.9 m 38
79 1.2 4.4 0.9 3.0 0.5 f 20
80 1.1 2.0 0.9 0.6 0.5 m 20
81 1.0 4.6 0.7 3.45 0.5 f 21
82 1.0 3.8 1.2 2.15 0.5 f 22
83 1.6 4.6 1.0 2.9 0.7 m 29
84 2.6 3.8 0.3 2.3 1.2 m 39
85 1.1 4.5 1.0 3.0 0.5 f 22
86 1.0 5.2 1.2 3.6 0.5 m 20
87 1.0 4.5 1.0 3.1 0.5 f 20
88 1.1 3.8 1.0 2.3 0.5 m 21
89 1.2 5.9 1.2 4.2 0.5 f 21
90 1.1 3.8 1.2 2.1 0.5 m 20
91 1.1 6.4 1.4 4.5 0.5 f 21
92 1.3 4.3 1.4 2.4 0.6 f 25
93 1.4 4.5 1.2 2.7 0.6 f 28
94 1.0 6.3 1.2 4.7 0.6 f 23
95 1.2 5.7 1.4 3.8 0.5 m 20
79
96 1.4 5.0 1.4 3.0 0.6 f 29
97 1.4 4.3 1.2 2.5 0.6 f 26
98 1.0 5.0 1.2 3.4 0.5 f 22
99 2.4 5.0 1.4 2.6 1.1 f 37
100 1.5 4.3 1.2 2.4 0.7 f 33
101 3.1 2.8 0.5 0.9 1.4 f 38
102 0.9 6.6 2.5 3.7 0.4 m 20
103 1.4 3.6 1.4 1.6 0.6 m 28
104 1.6 4.2 1.5 2.0 0.7 m 28
105 1.2 5.2 2.2 2.5 0.5 m 21
106 1.2 3.6 1.1 2.0 0.5 f 22
107 1.8 4.2 1.0 2.4 0.8 f 30
108 1.1 4.6 1.7 2.4 0.5 m 20
109 1.2 3.2 1.2 1.5 0.5 m 21
110 1.6 3.8 1.2 1.9 0.7 f 30
111 1.0 5.2 1.2 3.6 0.5 m 20
112 1.0 4.5 1.0 3.1 0.5 f 22
113 1.2 5.9 1.2 4.2 0.5 f 20
114 1.1 6.4 1.4 4.5 0.5 f 21
115 1.3 4.3 1.4 2.4 0.6 f 26
116 1.2 5.7 1.4 3.8 0.5 m 22
117 1.4 5.0 1.4 3.0 0.6 f 26
118 1.4 4.3 1.2 2.5 0.6 f 28
119 1.0 5.0 1.2 3.4 0.5 f 20
120 2.4 5.0 1.4 2.6 1.1 f 39
80
121 1.5 4.9 0.98 3.39 0.66 m 36
122 1.21 4.93 1.17 3.21 0.55 m 22
123 1.21 3.56 1.05 1.96 0.55 f 20
124 1.13 4.52 0.86 3.15 0.51 f 20
125 1.45 3.83 1.30 1.87 0.66 f 25
126 1.37 3.83 0.62 2.59 0.62 m 38
127 1.37 3.01 0.56 1.83 0.62 m 40
128 1.13 3.28 0.86 1.91 0.51 m 25
129 1.04 4.38 1.42 2.49 0.47 f 20
130 1.37 4.65 1.48 2.55 0.62 f 36
131 1.37 4.11 1.05 2.44 0.62 m 38
132 1.08 3.28 1.02 1.77 0.49 f 22
133 1.45 3.56 1.17 1.73 0.66 f 36
134 1.13 3.83 0.80 2.52 0.51 f 38
135 1.33 5.2 1.55 3.05 0.60 f 36
136 1.93 3.28 0.49 1.91 0.88 f 40
137 1.53 4.11 1.24 2.17 0.70 f 36
138 1.69 3.83 0.93 2.13 0.77 m 38
139 1.13 4.65 1.42 2.72 0.51 f 23
140 1.29 3.28 1.48 1.21 0.59 m 22
141 1.45 4.11 1.67 1.78 0.66 f 25
142 1.29 5.2 1.48 3.13 0.59 f 24
143 1.21 4.93 0.98 3.4 0.55 f 21
144 1.53 4.65 0.62 3.33 0.70 f 28
145 1.45 3.83 0.98 2.19 0.66 m 28
81
146 3.3 3.3 0.4 1.4 1.5 f 33
147 0.9 6.1 1.6 4.1 0.4 m 20
148 1.0 4.3 1.9 1.9 0.5 f 20
149 1.1 4.1 1.4 2.2 0.5 f 22
150 1.1 4.1 1.6 2.0 0.5 f 22
151 1.5 5.2 1.3 3.2 0.7 m 28
152 1.1 4.6 1.7 2.4 0.5 f 21
153 2.3 3.3 0.5 1.7 1.1 f 38
154 1.5 5.6 1.3 3.6 0.7 m 32
155 1.6 4.8 0.7 3.4 0.7 f 30
156 1.2 5.0 1.6 2.8 0.6 f 24
157 1.0 4.6 1.3 2.8 0.5 f 22
158 3.1 11.7 1.4 8.9 1.4 m 38
159 1.1 4.8 0.9 3.4 0.5 f 22
160 1.2 4.8 1.1 3.2 0.5 f 20
161 1.0 5.6 1.5 3.6 0.5 f 21
162 1.5 5.4 1.5 3.2 0.7 m 30
163 1.5 5.0 1.5 2.8 0.7 m 34
164 1.1 4.4 1.2 2.7 0.5 f 22
165 1.4 3.5 0.6 2.3 0.6 f 28
166 1.0 5.0 1.4 3.1 0.5 m 22
167 1.1 5.0 1.4 3.1 0.5 m 20
168 1.8 4.8 1.5 2.5 0.8 f 26
169 1.0 6.2 1.7 4.0 0.5 m 21
170 1.6 3.7 0.8 2.2 0.7 f 26
82
171 1.1 4.8 1.1 3.2 0.5 m 22
172 1.0 4.2 1.0 2.7 0.5 f 20
173 1.1 2.8 0.7 1.6 0.5 m 24
174 1.6 6.8 0.9 5.2 0.7 m 26
175 1.6 3.2 0.7 1.8 0.7 m 28
176 1.3 5.2 1.8 2.8 0.6 m 22
177 1.2 5.0 1.5 3.0 0.5 f 20
178 1.1 5.4 1.2 3.7 0.5 m 21
179 2.4 4.2 2.0 1.1 1.1 f 38
180 1.2 4.8 1.6 2.7 0.5 m 22
181 2.2 5.6 1.4 3.2 1.0 f 39
182 2.4 5.4 1.1 3.2 1.1 f 40
183 1.3 4.6 0.9 3.1 0.6 f 24
184 1.4 3.4 0.6 2.2 0.6 f 28
185 1.3 5.2 1.4 3.2 0.6 m 24
186 1.3 4.6 1.3 2.7 0.6 m 26
187 1.1 5.0 1.3 3.2 0.5 m 20
188 2.0 3.0 0.8 1.3 0.9 m 36
189 1.2 4.4 0.9 3.0 0.5 f 20
190 1.1 2.0 0.9 0.6 0.5 m 21
191 1.0 4.6 0.7 3.45 0.5 f 22
192 1.0 3.8 1.2 2.15 0.5 f 24
193 1.6 4.6 1.0 2.9 0.7 m 29
194 2.6 3.8 0.3 2.32 1.2 m 35
195 1.1 4.5 1.0 3.0 0.5 f 20
83
196 1.1 3.8 1.0 2.3 0.5 f 22
197 1.1 3.8 1.2 2.1 0.5 m 22
198 1.4 4.5 1.2 2.7 0.6 f 26
199 1.0 6.3 1.2 4.7 0.5 f 21
200 1.5 4.3 1.2 2.43 0.7 f 28
MALE AND FEMALE CONTROL SAMPLE RESULT.
S/NO TG TC HDL LDL VLDL SEX AGE(yrs)
1 1.0 3.2 1.0 1.75 0.45 m 20
2 1.1 4.6 1.2 2.9 0.5 m 25
3 1.5 2.6 0.6 1.32 0.68 m 29
4 1.0 3.6 1.3 1.85 0.45 m 30
5 1.1 3.8 1.3 2.0 0.5 m 22
6 1.0 4.2 1.4 2.35 0.45 f 24
7 2.2 3.4 0.4 2.0 1.0 f 40
8 1.7 4.0 1.2 2.03 0.77 f 36
9 0.8 3.8 1.5 1.94 0.36 f 20
10 1.3 6.0 1.5 3.91 0.59 f 24
11 1.3 5.2 1.2 3.41 0.59 f 26
12 1.4 4.6 1.1 2.9 0.6 m 28
13 1.7 4.4 1.4 2.23 0.77 m 28
14 1.3 6.1 1.4 4.15 0.59 f 22
15 1.0 6.4 1.5 4.42 0.45 f 20
16 1.6 4.5 1.2 2.61 0.7 f 22
17 1.3 5.2 1.2 3.42 0.59 m 21
84
18 1.7 3.8 1.4 1.67 0.77 f 30
19 1.3 5.0 1.5 2.9 0.6 f 20
20 1.4 3.5 1.2 1.7 0.6 m 26
21 1.6 5.0 1.4 2.9 0.7 f 28
22 1.2 5.0 1.5 3.0 0.5 m 22
23 1.6 4.5 1.4 2.4 0.7 f 26
24 1.3 4.7 1.5 2.6 0.6 f 24
25 1.3 4.4 1.2 2.6 0.6 m 26
26 1.3 6.1 1.36 4.15 0.59 f 25
27 1.0 6.4 1.53 4.42 0.45 f 20
28 1.6 4.5 1.19 2.61 0.7 f 28
29 1.3 5.2 1.19 3.42 0.59 f 22
30 1.7 3.8 1.36 1.67 0.77 f 30
31 1.0 3.2 1.0 1.75 0.45 m 20
32 1.1 4.6 1.2 2.9 0.5 m 24
33 1.5 2.6 0.6 1.32 0.68 m 38
34 1.0 3.6 1.3 1.85 0.45 m 22
35 1.1 3.8 1.3 2.0 0.5 m 20
36 1.0 4.2 1.4 2.35 0.45 m 21
37 2.2 3.4 0.4 2.0 1.0 m 39
38 1.7 4.0 1.2 2.03 0.77 m 32
39 0.8 3.8 1.5 1.94 0.36 m 21
40 1.3 6.0 1.5 3.91 0.59 m 26
41 1.3 5.2 1.2 3.41 0.59 f 24
42 1.4 4.6 1.1 2.9 0.6 f 28
85
43 1.7 4.4 1.4 2.23 0.77 m 32
44 1.3 6.1 1.36 4.15 0.59 f 26
45 1.0 6.4 1.53 4.42 0.45 f 24
46 1.6 4.5 1.19 2.61 0.7 f 25
47 1.3 5.2 1.19 3.42 0.59 f 22
48 1.7 3.8 1.36 1.67 0.77 f 36
49 1.0 3.2 1.0 1.75 0.45 m 22
50 1.1 4.6 1.2 2.9 0.5 m 20
51 1.5 2.6 0.6 1.32 0.68 m 28
52 1.0 3.6 1.3 1.85 0.45 m 21
53 1.1 3.8 1.3 2.0 0.5 m 22
54 1.0 4.2 1.4 2.35 0.45 m 24
55 2.2 3.4 0.4 2.0 1.0 m 36
56 1.7 4.0 1.2 2.03 0.77 m 30
57 0.8 3.8 1.5 1.94 0.36 m 22
58 1.3 6.0 1.5 3.91 0.59 m 22
59 1.3 5.2 1.2 3.41 0.59 f 24
60 1.4 4.6 1.1 2.9 0.6 f 24
61 1.7 4.4 1.4 2.23 0.77 f 36
62 1.0 3.2 1.0 1.75 0.45 m 24
63 1.1 4.6 1.2 2.9 0.5 m 20
64 1.5 2.6 0.6 1.32 0.68 m 26
65 1.0 3.6 1.3 1.85 0.45 m 22
66 1.1 3.8 1.3 2.0 0.5 m 24
67 1.0 4.2 1.4 2.35 0.45 f 20
86
68 2.2 3.4 0.4 2.0 1.0 f 32
69 1.7 4.0 1.2 2.03 0.77 f 36
70 0.8 3.8 1.5 1.94 0.36 f 21
71 1.3 6.0 1.5 3.91 0.59 f 22
72 1.3 5.2 1.2 3.41 0.59 f 24
73 1.4 4.6 1.1 2.9 0.6 m 28
74 1.7 4.4 1.4 2.23 0.77 m 26
75 1.3 6.1 1.4 4.15 0.59 f 22
76 1.0 6.4 1.5 4.42 0.45 f 20
77 1.6 4.5 1.2 2.61 0.7 f 27
78 1.3 5.2 1.2 3.42 0.59 f 21
79 1.7 3.8 1.4 1.67 0.77 f 28
80 1.3 5.0 1.5 2.9 0.6 m 20
81 1.4 3.5 1.2 1.7 0.6 m 26
82 1.6 5.0 1.4 2.9 0.7 f 28
83 1.2 5.0 1.5 3.0 0.5 m 22
84 1.6 4.5 1.4 2.4 0.7 f 28
85 1.3 4.7 1.5 2.6 0.6 f 22
86 1.3 4.5 1.2 2.6 0.6 m 20
87 1.5 5.6 1.3 3.6 0.7 m 24
88 0.9 6.6 2.5 3.7 0.4 m 21
89 1.0 5.6 1.5 3.6 0.5 m 24
90 1.5 5.4 1.5 3.2 0.7 m 24
91 1.0 5.0 1.4 3.1 0.5 m 22
92 1.1 5.0 1.4 3.1 0.5 m 20
87
93 1.3 4.6 0.9 3.1 0.6 f 22
94 1.4 3.4 0.6 2.2 0.6 f 26
95 1.6 3.8 1.2 1.9 0.7 f 28
96 1.1 5.0 1.3 3.2 0.5 f 24
97 1.2 4.4 0.9 3.0 0.5 f 22
98 1.0 4.6 0.7 3.5 0.5 f 20
99 1.0 3.8 1.2 2.2 0.5 f 22
100 1.3 4.3 1.4 2.4 0.6 f 21
88
APPENDIX 11
FORMULAE USED IN STATISTICAL ANALYSIS
1. Mean(X)
(X) = x
n where x = sum of values obtained
n = number of values obtained
X = mean of values obtained
2. Standard deviation (S)
S = (x-x)2 n- 1 where x =values obtained
X= mean of values obtained
n= Number of values obtained
3. Student’s t – distribution (t – dist).
t – dist = x1 – x2
SD21 + SD2
2 n1 n2
Where x1 = Means of volume obtained for test subject
x2 = Means of values obtained for control
SD1 = Standard deviation of test subject
SD2 = Standard deviation of control
n1 = Number of values obtained for test subject
n2 = Number of values obtained for control
This formulae is basically used to determine whether there is a
significant difference between, means of two different group of
samples.
89
4. CORRELATION
This was done using computer with the method named graphed
prism soft ware. Also this formulae was used.
correlation coefficient (r) = (x1 – x1) (x2 – x2)
[ (x – x) (x –x) ]
Where X1 = Values obtained for test subjects
X2 = Values obtained for controls
X1 = Mean value for the test subjects
X2 = Mean value for the control
Then, t = r n – 2
1 – r2
With a degree of freedom of n – 2
Where n = number of values obtained.
5. One way Analysis of variance (ANOVA)
The method is useful for comparing the means of
measurements made on 3 or more groups.
The analysis is carried out by
(i) Calculating between group sum of squares given by
E x 2 - E x 2 n N
(ii) Calculating within group sum of squares given by
E x2 - E x 2
n (iii) Calculating the degree of freedom for between groups given by k
– 1 where k represents number of groups.
(iv) Calculating the degree of freedom for within groups given by
N - k
90
Where N represents the total number of subjects and k
represents the number of groups.
F ratio = Ex 2 - E x 2
n N
Ex2 - Ex 2
n
APPENDIX 111
All the reagents for lipids (Triglyceride and cholesterol) assay were
obtained from biosystems S.A. costa Brava, 30, Barcelona Spain.
1. Triglyceride Reagent
Composition
A. Reagent : Pipes 45 mmol/L, magnesium chloride 5mmol/L, 4-
chlorophenol 6mmol/L, lipase > 100µ/ml, glycerol kinase > 1.5
µ/ml, glycerol – 3-phosphate oxidase > 4µ/ml, peroxidase > 0.8
µ/ml, 4 – aminoantipyrine 0.75mmol/l, ATP 0.9mm0l/L, PH 7.0
S. Triglycerides standard: Glycerol with surfactant equivalent to
200mg/100ml (2.26mmol/L) triolein and sodium azide 0.1% as
preservative.
Reagent preparation
Reagents and standard are provided ready to use
2. Cholesterol Reagent
Composition
A. Reagent. Pipes 35mmol/L, sodium cholate 0.5mmol/L, phenol
28mmol/L, cholesterol esterase > 0.2µ/ml, cholesterol oxidase >
0.1µ/ml, peroxidase > 0.8µ/ml, 4–amino antipyrine 0.5mmol/L,
PH 7.0
91
S. Cholesterol standard cholesterol 200mg/dl, (5.18mmol/L) Aqueous
primary standard.
Reagent preparation
Reagent and standard are provided ready to use,
3. Cholesterol HDL Reagent
Contents and composition.
A. Reagent: 2 x 50mL. phosphotungstate 0.4mmol/L,
magnesium chloride 20mmol/L.
B. Reagent: 2 x 50ml. phosphate 35mmol/L, cholesterol esterase >
0.2µ/ml, cholesterol oxidase > 0.1µ/mL, peroxidase > 1µ/ml, 4
– aminoantipyrine 0.5mmol/L, sodium cholate 0.5mmol/L,
dichlorophenol sulfonate 4mmol/L, PH 7.0
S. HDL Cholesterol standard: 1 x 5ml. cholesterol 15mg/dL
Aqueous primary standard.
Reagent preparation
Reagents and standard are provided ready to use.
92
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