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

REFERENCES

Abuobeida A.A.A (1996): Typhoid and paratyphoid fever, Africa

Health, 18 (5):14-15.

Aduba O, Onwuamaeze I, Oli J, Udeozo K (1984): Serum cholesterol

and HDL cholesterol in Nigeria diabetics. East Africa Medical

Journal, 61:35-39.

Agomuoh D.I, (2006): Lipid profile of Healthy adult Nigerians in

Portharcourt, Nigeria; Nigeria Journal of Medicine 15(2)

137-140.

Ajose O.A, Fasubaa,O.B, Thomas, K.D. Bolodeoku, J.O.(2002).

Serum lipids and lipoprotein cholesterol profile in pregnant

Nigerian women. Journal of clinical Science 2(1-2)9-13.

Allain C.C,Poon LS, Chan CSG, Richmond W,FU P.C (1974):

Enzymatic determination of total serum cholesterol. Clinical

chemistry; 20:470-475

Animashaun T, Odugbemi.T(1990) Widal Test: Its use and abuse in

Nigeria, Nigeria Medical Practitioner, 19: 96- 98.

Bales A. C (2000): In search of lipid balance in older women;

New studies raise questions about what works best.

Postgraduate Medicine 108(7):57-72.

Baron D N (1988): Lipid metabolism. A short textbook of chemical

pathology, 4th edition ELBB, London, 76-88.

Braunwald E, Fauci A, Kasper D, Hauser S, Longo D, Jameson

J,(2001); Disorders of lipoprotein metabolism: Harrisons

70

Principles of Internal Medicine 15th edition, McGraw-Hill

New york,2247-2257.

Burtis C.A; Ashwood. E.R(1999);Lipids, Lipoproteins, and

Apolipoproteins: Tietz textbook of clinical chemistry, 3rd

edition, W.B. Saunders Company, United States of

American; 809-861.

Burtis C.A; Ashwood .E.R.(1999); Lipids Lipoproteins, and

Apolipoproteins; Fundamentals of clinical chemistry 5th

edition,463-493.

Cooper G R (1982): Distribution of total cholesterol values in the U.S.

Population. In disorder of lipid metabolism. Clinical chemistry-

theory, analysis and correlation 2nd edition, Kaplan L A, Pesce A

J, editors, C V mosby company, Toronto 454-483.

Delvin T. M, (1993); Lipids and lipoproteins: Textbook of biochemistry

with clinical correlations 3rd edition, Wiley-loss publication New

York 387-417.

Ehnholm C, Lukka M, Kuusi T, Nikkila E, Utermann G (1986):

Apolipoprotein E polymorphism in the finnish population, gene

frequencies and relation to lipoprotein concentration J lipid Res.

27:227-235.

Fossati P, Prencipe L. (1982): Serum Triglycerides determined

colorimetrically with an enzyme that produces hydrogen

peroxidase. Clinical chemistry; 28 (10):2077 – 2080.

71

Friedewald W.J. Levy R.I, Fredrickson DS (1972): Estimation of

the concentration of low density lipoprotein – cholesterol in

plasma without use of the preparative ultracentrifuge.

Clinical Chemistry, 18:4599- 502.

Graham S. M. and Graham S.M. (2002): Salmonellosis in children

in developing and developed countries and populations:

Current opinion in infections Diseases; 15 (5): 507 – 512.

Grande F (1982): Diets of different fatty acid composition producing

identity serum cholesterol levels in man. American Journal of

clinical Nutrition 34:2111-2119.

Grundy S.M. (1999) Primary Prevention of coronary heart disease:

Integrating risk assessment with intervention circulation 100:

988.

Hatta M. (2002) Simple dipstick assay for the detection of

salmonella typhi-specific 1gM antibodies and the evolution of

the immure response in patients with typhoid fever. American

Journal of Tropical Medicine. 66(4): 416 – 421.

House Deborah, Wain John, HO VO A. Diep TO S; Chinh Nguyen,

Bay Phan V, Vinh Ha, Due Minh, Parry Christopher M,

Dougan Gordan, white Nicholas J, Hien Trren Tinh and Farrar

Jeremy J. (2001); Typhoid Fever complications: Journal of clinical

microbiology, 39 (3) 1002 – 1007.

72

How is typhoid fever diagnosed (2006) http://www.doctorndtv.

Com/FAQ/detailfaq.asp?id.

Http.///www.health a to z. com/health a to z/ A to Z /ency/typhoid

fever. Jsp (2005).

Http.///www.hap.org/healthy living / lipid profile. Php (2004).

Igweh. J.C. Aloamaka, C.P. (2003) cholesterol profile of Adults

resident in Eastern Nigeria O.J Med 15 (3 & 4) 46 – 50.

Ira S.M. and Wolinsky F.E. (1998): Lipid metabolism. Nutrition in

exercise and sport, 3rd edition W.B. Saunders Company, 105 –

110.

Katung P. Yaya (2000): A brief review of typhoid fever in Nigeria,

Nigeria Medical Practitioner 38: 4 -6

Khosla S.N, Goyle N, Seth RK (1991): Lipid profile in enteric fever;

Journal association of India physicians; 39 (3) 260 – 262.

Lipid profile (2004): Http://www.sgrh.com/faq/lipid. htm.

Meaning of Enteric fever (2004): http://www.hyper-

dictionary.com/dictoinary/enteric+fever.

Murray R.K. Granner D.K. Hayes P.A, Rodwell V.W. (2000): Lipids of

physiologic significance; harpers Biochemistry 25th edition,

MC Grawhill, London: 160 – 172.

National Cholesterol Education Program Expert Panel. (2001): Third

report of the National Cholesterol Education Program (NCEP)

Expert Panel on detection, Evaluation and Treatment of

High Blood Cholesterol in Adults (ATPIII). NIH Publication.

Bethesda: National Heart, Lung and Blood Institute.

73

National Cholesterol Education Programme, NCEP (1993):

summary of the second report of the National cholesterol

education program (NCEP) Expert panel on detection in adults –

JAMA, 269 (23): 3015 – 3023.

Naito HK (1989b): Disorders of Lipid metabolism; clinical

Chemistry, Theory, Analysis and Correlation 2nd edition, Kaplan

LA, Pesce AJ, editors, CV Mosby Company. Toronto: 454 –

483.

Ohio Health (2004): Understanding your lipid profile; nutrition and

weight management, MC Connell Heart Health Center; 1:1

Ozumba N.A. Ozumba U.C. Obionu C; Nwakoby B.A. N; amah

Angela and Ugochukwu C. (1997): Typhoid fever epidemic in

Nigeria; Journal of College of Medicine 2 (2): 31 – 33.

Paratyphoid Fever Http://www.health a to z. com/health a to Z/A

ency/paratyphoid fever (2006).

Richens J. (2004): Typhoid fever: Infectious diseases in Cohen J

and Powderly Editors; Mosby Inc; Edinburgh. 1561 – 1565.

Schaefer E.J. Lamon Fava S, Colin SD, Schaefer MM, Ordovas J.M,

Castelli Wp. Wilson PWF. (1994): Effects of age, gender and

menopausal status on plasma low density-lipoprotein

cholesterol and apolipoprotein B levels in the framinghan

offspring study: Journal of lipid Research 35: 779 – 792.

Segrest, J.P. (2001): Structure of apolipoprotein B – 100 in low-

density lipoprotein: Journal of lipid Research 42: 1346 – 1367

74

Thielman N.M. (2005): Enteric fever: Principles and Practice of

Infectious Diseases. Mandell, Bennett and Dolin. Philadelphia.

Churchill living stone; 1273 – 1279.

Tikkanen M J, Huttunen J K, Ehnholm C, Pietinen P (1990):

Apolipoprotein E4 homozygosity predisposes to serum

cholesterol elevation during high fat diet. Arteriosclerosis 10(2):

285-288.

Understanding your lipid profile (2004): Http://www.ohiohealth.

Com/facilities/MCconell/weight

manage/details/understand lipid.

Vartianen E, puska p, Salomen J T (1982): serum total cholesterol,

HDL-cholesterol and blood pressure levels in 13 year old

children in Eastern firland. Acta Med Scand, 211;95-102.

Wikimedia Foundation (2007): Apolipoprotein; wikipedia

encyclopedia, 17:23.

Wooton IDP, Freeman H (1982): Blood lipids microanalysis in medical

Biochemistry 6th edition, Churchill livingstone,167-177.

Zenilman, J.M. (1997): Typhoid Fever; Journal of the American

medical Association (1): 847 – 850

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