RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1
Name of the Candidate and Address
RACHAEL LALMALSAWMKIMI
St. John’s Medical College
Sarjapur Road
Koramangala
Bangalore – 560 034
2
Name of the Institution
St. John’s Medical College
Bangalore – 560 034
3
Course of study and subject
4
Date of Admission to course
5
TITLE OF THE TOPIC
Title:
MSc. MLT ( Biochemistry)
September 2010
Evaluation of Serum Albumin in individuals having
Hyperlipidemia.
6
BRIEF RESUME OF THE INTENDED WORK
6.1 Need of the Study:
Hyperlipidemia occurs when a person’s cholesterol (or) triglycerides is raised to
levels beyond what is considered normally safe. There is a hereditary factor of the
disease as well as factors caused by the diet and exercise choices of the person
suffering from the disease.
Hyperlipidemia is a risk factor for heart disease. A wide variety of
epidemiological data indicates that hyperlipidema is a predictor of coronary heart
disease (CHD) 1.
Various studies have reported an inverse association between serum albumin level
and incident of CHD, though the exact biological mechanisms have not been
established. Proposed mechanism (or) confounding factors may include:
1. Albumin showing correlation with inflammation and infection.2
This correlation may be attributed to chronic inflammatory disease, because
several studies demonstrated association between serum albumin and
inflammatory markers.3,4,5
2. Its relation to fibrinolysis and hemostasis factors.3
3. Possible role in platelet aggregation.4
4. Its role as an antioxidant.5
5. Its possible role as marker of increased vascular permeability due to
underlying disease.2,6
6. Its relation to nutritional status.7
The present study is undertaken to evaluate the
relationship between serum
albumin and hyperlipidemia which is a potential risk factor for cardiovascular
disease.8
6.2 Review Of Literature
ALBUMIN
Albumin is a hepatic protein, and its plasma concentration is controlled by several
factors, including rate of albumin synthesis, catabolic rate, albumin distribution
and exogenous albumin loss.10 Synthesis of albumin is affected by nutritional
intake, colloidal osmotic pressure variations and the presence of systemic
inflammation.10,11 The primary function of albumin is generally considered to be
the maintenance of colloidal osmotic pressure (COP) in both the vascular and
extravascular spaces. Albumin is essential for the metabolism and detoxification
of free fatty acids, phospholipids, cholesterol, amino acids, drugs and many
others.
Serum albumin is the most abundant plasma protein. High levels of
albumin are partially protective against artherosclerosis, probably because of its
promotion of cholesterol efflux from fibroblast and other cells.12,13
Reference Intervals: Based on the International Protein Reference CRM 470, the
recommended interim reference interval for albumin in serum of adults 20 – 40
yrs of age is 35 – 52 g/L (3.5 – 5.2 g/dl).9
LIPID PROFILE
Cholesterol is the precursor of all steroid hormones and of the bile salts which are
essential for the digestion and absorption of lipids. Increased cholesterol is a risk
factor in the cause of atherosclerotic disease. Further studies showed that when the
total cholesterol concentration is high, the incidence and prevalence of CHD are
also high. Many epidemiological and clinical studies have shown that both
increased LDL cholesterol and increased HDL cholesterol are associated with an
increased risk of CHD.13
Reference range of lipid profile 21
Total cholesterol < 200 mg/dl
LDL cholesterol <130 mg/dl
HDL cholesterol
Triglyceride
40-60 mg/dl
< 150 mg/dl
Hyperlipidemia
Hyperlipidemia occurs when a person’s cholesterol or triglycerides is raised to
levels beyond what is considered normally safe. There is a hereditary factor of the
disease as well as factors caused by the diet and exercise choices of the person
suffering from the disease. Hyperlipidemia is a risk factor for coronary artery
disease .
Several prospective studies have demonstrated an association between low serum
albumin and increased cardiovascular morbidity and mortality.2,14,19,20 A reduction
in serum albumin over time is associated with increased incidence of
cardiovascular disease, even if the change is within the normal albumin range. 15
Low serum albumin may be a reflection of inflammatory burden predisposing to
CHD.10,16 Measurement of serum albumin is inexpensive, can be used as a tool to
identify individuals at risk for CHD.17
6.3 Aims And Objectives Of The Study:
 Estimation of serum Lipid profile- Total cholesterol, LDL, and HDL
cholesterol.
 Estimation of serum albumin.
 Correlate the values of serum albumin with Lipid profile.
7
MATERIALS AND METHODS
7.1 SOURCE OF DATA:
SUBJECTS & CONTROL :
 Age- 25 – 55 years old
 Males and females coming to St. John’s medical college & hospital
laboratory for testing fasting Lipid profile.
 Sample size : 50.
Control : 25
Subject : 25
7.2
Inclusion Criteria
 25–55 yrs, Males and Females.
 Family history of heart disease
 Family history of diabetes
 History of smoking
 History of Alcoholism
7.3
Exclusion Criteria
 Males and females less than 25yrs and more than 55yrs
 History of chronic liver disease
 History of chronic renal disease
 Pregnant women
7.4 Methods
 A questionnaire will be prepared containing questions regarding inclusion
and exclusion criteria.
 Questionnaire will be given to the patients coming to St. John’s Hospital
Laboratory for providing fasting blood samples for Lipid profile.
 5ml of blood will be collected in a vacutainer and sent to the Biochemistry
laboratory for analysis of Lipid profile by autoanalyser.
 The same sample will be utilized for the estimation of serum albumin by
BCG method manually.
 Subjects and controls will be selected according to the Lipid parameters.
Subjects : abnormal lipid profile
Controls : normal lipid profile
 Correlation of serum albumin values with lipid parameters of both control
and subjects will be done.
ESTIMATION OF SERUM ALBUMIN
NAME OF THE METHOD: BCG manual method 18
PRINCIPLE: At a pH lower than its isoelectric point, albumin is positivey charged
and has an affinity for anions. The anionic dyes Bromocresol green (BCG)
combine with albumin to form a bluish-green complex. The intensity of colour
produced is directly proportional to the concentration of albumin present in the
sample.
REAGENTS:
1. Citrate buffer : Prepared by dissolving 6.2g of citric acid and 6g of
trisodium citrate in 500ml of distilled water. The ph is adjusted to 4.
2. Stock BCG: Prepared by dissolving 210mg of BCG and 5ml of 0.1N NaOH
and the volume is made upto 500ml with distilled water.
3. Working BCG: Prepared by dissolving 125ml of stock solution in 375ml of
citrate buffer. To this, 2ml of Trition-10 is added.
PROCEDURE:
Clean test tubes labeled as Blank (B), Standard (S), and Test (T).
Reagents
B
Working standard (ml)
0.9% NaCl (ml)
S1
S2
S3
S4
S5
T
0 0.02
0.04
0.06
0.06
0.1
-
0.1 0.08
0.06
0.04
0.04
0.0
-
Conc of albumin in glloo ml
0
1.5
3.0
4.5
4.5
7.5
-
Serum (ml)
-
-
-
-
-
-
0.1
4.9
4.9
4.9
4.9
4.9
4.9
4.9
BCG reagent (ml)
Mix well; incubate at room temperature for 30mins or for 10mins in an incubator.
Read the Absorbance at 630nm.
CALCULATIONS:
Albumin (g/dl) = T – B x Conc. of albumin in Standard (g/dl)
S–B
ESTIMATION OF LIPID PROFILE
TRIGLYCERIDES
Name of method: Enzymatic method (LPL/GK/GPO/POD)
Principles of procedures: The triglycerides method is based on an enzymatic
procedure in which combination of enzymes are employed for the measurement of
serum or plasma triglycerides. The sample is incubated with lipoprotein lipase
(LPL) enzyme reagent that converts triglycerides into free glycerol and fatty
acids. Glycerol kinase (GK) catalyzes the phosphorylation of glycerol by
adenosine-5-triphosphate (ATP) to glycerol-3-phosphate. Glycerol-3-phosphateoxidase oxidizes glycerol-3-phosphate to dihydroxyacetone phosphate and
hydrogen peroxide (H2O2). The catalytic action of peroxidase (POD) forms
quinoneimine from H2O2, aminoantipyrine and 4-chlorophenol. The change in
absorbance due to the formation of quinoneimine is directly proportional to the
total amount of glycerol and its precursors in the sample and is measured using a
bichromatic (510, 700nm) endpoint technique.
LPL
Triglycerides
Glycerol + Fatty acids
GK
Glycerol + ATP
Glycerol-3-phosphate + ADP
GPO
Glycerol-3-phosphate + O2
Dihydroxyacetone phosphate + H2O2
POD
2 H2O2 + Aminoantipyrine
+ 4-Chlorophenol
Quinoneimine + HCL + 4 H2O
TOTAL CHOLESTEROL
Name of method: Enzymatic method (CE/CO/HPO)
Principles of procedures: Cholesterol esterase (CE) catalyzes the hydrolysis of
cholesterol esters to produce free cholesterol which, along with preexisting free
cholesterol, is oxidized in a reaction catalyzed by cholesterol oxidase (CO) to
form cholest-4-ene-3-one and hydrogen peroxide. In the presence of horseradish
peroxidase (HPO), the hydrogen peroxide thus formed is used to oxidize N, N
diethyaniline-HCl/4-aminoantipyrine (DEA-HCl/AAP) to produce a chromophore
that absorbs at 540nm. The absorbance due to oxidized DEA-HCl/AAP is directly
proportional to the total cholesterol concentration and is measured using a
polychromatic (452, 540, 700nm) endpoint technique.
CE
Cholesterol esters
Cholesterol + Fatty acids
CO
Cholesterol + O2
Cholest-4-ene-3-one + H2O2
HPO
2H2O2 + DEA-HCl/AAP
4 H2O + Oxidised DEA-HCl/AAP
HIGH DENSITY LIPOPROTEIN (HDL)
Methodology: Accelerator selective detergent.
Principles of procedures: The HDL cholesterol assay is a homogenous method for
directly measuring HDL-C levels without the need for offline pretreatment or
centrifugation steps.
The method is in a two reagent format and depends on the properties of a unique
detergent, as illustrated. This method is based on accelerating the reaction of
cholesterol oxidase (CO) with non-HDL unesterified cholesterol and dissolving
HDL selectively using a specific detergent.
In the first reagent, non-HDL unesterified cholesterol is subject to an enzyme
reaction and the peroxidase generated is consumed by a peroxidase reaction with
DSBmT yielding a colorless product. The second reagent consists of a detergent
capable of solubilizing HDL specifically, cholesterol esterase (CE) and
chromogenic coupler to develop color for the quantitative determination of HDLC. This may be referred to as the Accelerator Selective Detergent methodology.
Accelerator selective Detergent Methodology
Accelerator + CO
HDL, LDL, VLDL, Chylomicrons
Non-reactive LDL, VLDL,
DSBmT + Peroxidase
Chylomicrons
HDL Specific Detergent
HDL
HDL disrupted
Cholesterol esterase
HDL Cholesterol
∆4 Cholestenone + H2O2
Cholesterol oxidase
Peroxidase
H2O2 + DSBmT + 4-AAP
Color development
LOW DENSITY LIPOPROTEIN (LDL)
Methodology : Detergent/CE/CO/POD
Principles of procedures: The LDL cholesterol assay is a homogenous method for
directly measuring LDL-C levels in human serum or plasma, without the need for
any offline pretreatment or centrifugation steps. The method is in a two reagent
format and depends on the properties of detergent 1 which solubilizes only nonLDL particles. Cholesterol released is consumed by cholesterol esterase and
cholesterol oxidase in a non color forming reaction. Detergent 2 solubilizes the
remaining LDL particles.
The soluble LDL-C is then oxidosed by the action of cholesterol esterase and
cholesterol oxidase forming cholestenone and hydrogen peroxide (H2O2). The
enzymatic action of peroxidase on H2O2. The enzymatic action of peroxidase on
H2O2 produces color in the presence of N,N-bis(4-sulfobutyl)-m-toluidine,
disodium salt (DSBmT) and 4-aminoantipyrine (4-AA) that is measured using a
bichromatic (540, 700nm) endpoint technique. The color produced is directly
proportional to the amount of LDL-C present in the sample.
Detergent 1
Non-soluble LDL-C, VLDL-C,
HDL-C, Chylomicrons
DSBmT + Peroxidase
Soluble Non-LDL-C, (VLDL-C, HDL-C,
Chylomicrons)
Cholesterol esterase
Soluble Non-LDL-C
Non-color forming
Cholesterol oxidase
Detergent 2
Non-soluble LDL-C
Soluble LDL-C
Cholesterol esterase
Soluble LDL-C + O2
Cholestenon + H2O2
Cholesterol oxidase
Peroxidase
H2O2 + DSBmT + 4-AA
Color development
STATISTICS: Statistical analysis will be done by appropriate Standard statistical
software programme (SPSS16).
METHOD: Correlation co-efficient.
7.5 Does the study require any investigations or interventions to be conducted
on patients or other humans or animals? If so, please described briefly.
No.
7.6 Has ethical clearance been obtained from your in case of 7.5
Not applicable.
8
LIST OF REFERENCES
1. Amin A Nanji, Suseela Reddy. Use of total cholesterol/albumin ratio as an alternative to
high density lipoprotein cholesterol measurement. J Clin Pathol 1983;36:716 – 718.
2. Kuller LH, Eichner JE, orchard TJ, et al. The relation between serum Albumin levels
and risk of coronary heart disease in the Multiple Risk Factor Intervention Trial Am J
Epidemiol 1991;134:1266-77.
3. Hunt BJ. The relation between abnormal hemostatic function and the progression of
coronary disease. Curr Opin Cardiol 1990;5:758-65.
4. Doweiko JP, Bistrain BR. The effect of glycosylated albumin on platelet aggregation. J
Prenter Enteral Nutr 1994;18:516-20.
5. Halliwell B. Albumin-an important extracellular antioxidant? Biochem Pharmcol
1988;37:569-71.
6. Fleck A, Raines G, Hawker F, et al. Increased vascular permeability: a major cause of
hypoalbuminaemia in disease and injury. Lancet 1985;1:781-4.
7. Gillum RF, Ingram DD, Makuc DM. Relation between serum albumin concentration
and stroke incidence and death: The NHANES I Epidemiologic Follow-up Study. Am J
Epidemiol 1994;140:876-88.
8. Nelson J.J.; Liao Duanping ; Sharrett et al. Serum Albumin Level as a Predictor of
Incident Coronary Heart Disease: The Atherosclerosis Risk in Communities (ARIC)
Study. Am J of Epidemiol 2000;151:468-477.
9. Carl A, Burtis Ph.D, Edward R. Ashwood M.D, David E.Bruns M.D. Teitz Textbook of
Clinical Chemistry and Molecular diagnostics; 4th Edition, 2006. Amino acids, peptides
and proteins. Chapter 23, pg: 549.
10. Don BR, Kaysen G. Serum albumin; relationship to inflammation and nutrition. Semin
Dial 2004;17:432-7.
11. Mitch WE, Malnutrition: a frequent misdiagnosis for hemodialysis patients. J Clin
Invest 2002;110:437-9.
12. Carl A, Burtis Ph.D, Edward R. Ashwood M.D, David E.Bruns M.D. Teitz Textbook of
Clinical Chemistry and Molecular diagnostics; 4th Edition, 2006. Lipids, Lipoproteins,
Apolipoproteins and other Cardiovascular Risk Factors. Chapter 26; 903-981.
13. Lehninger Principles of Biochemistry, 4th Edition. “Hormonal Regulation & Integration
of Mammalian metabolism” Chapter 23; pp.896.
14. Phillips A, Shaper AG, Whincup PH. Association between serum albumin and morality
from cardiovascular disease, cancer, and other causes. Lancet. 1989;2:1434-6.
15. Schalk BW, Visser M, Bremmer MA, et al. Changer of serum albumin and risk of
cardiovascular disease and all-cause mortality: Longitudinal Aging Study Amsterdam.
Am J Epidemiol 2006;164:969-77.
16. Cesari M, Penninx BW, Newman AB, et al. Inflammatory markers and onset of
cardiovascular events: results from the Health ABC study. Circulation 203;108:2317-22.
17. Deepa M. Gopal, MD, MS; Andreas P. Kalogeropoulos, et al. Serum Albumin
Concentration and Heart Failure Risk: The Health, Aging, and Body Composition
Study. Am Heart J 2010;160(2):279-285.
18. Alan H.Gowenlock. Plasma proteins; Varley’s Practical Clinical Biochemistry, 6th
edition 1996 pp 401 – 436.
19. Gillum RF, Makuc DM. Serum albumin, coronary heart disease, and death. Am Heart J
1992;123:507-13.
20. Klonoff-Cohen H, Barett-Connor EL, Edelstein SL. Albumin levels as a predictor of
mortality in the healthy elderly. J Clin Epidemiol 1992;45:207-12.
21. Carl A, Burtis Ph.D, Edward R. Ashwood M.D, David E.Bruns M.D. Teitz
fundamentals of clinical chemistry; 6th edition, 2008. Reference information for the
clinical laboratory. Chapter 45; pg: 836.
9
Signature of Candidate
10
Remarks of the Guide
11
Name and designation of :
12
11.1 Guide
: Dr. Radhika Krishnaswamy, MD, DNB.
Associate Professor,
Dept. of Biochemistry.
11.2 Signature
:
11.3 Co-guide
(If Any)
:
11.4 Signature
:
11.5 Head of the
Department
: Dr. Anitha R. Bijoor, MD
Professor & Head
Dept. of Biochemistry.
11.6 Signature
:
12.1 Remarks of the HOD :
12.2 Signature
QUESTIONNAIRE
Topic : Evaluation of Serum Albumin in individuals having Hyperlipidemia.
1. Name
:
2. Age (25-55)
:
3. Sex
:
4. IP/OPD No.
:
5. Department
:
6. History of
i) Diabetes mellitus
Yes
No
ii) Hypertension
Yes
No
iii) Chronic Renal failure / Dialysis
Yes
No
Yes
No
Yes
No
i) History of smoking
Yes
No
ii) History of Alcoholism
Yes
No
i) Diabetes mellitus
Yes
No
ii) Hypertension
Yes
No
iii) Cardiac diseases
Yes
No
iv) Myocardial Infarction
Yes
No
iv) Chronic Liver Diseases
v) Pregnancy
7. Personal History
8. Family History
VOLUNTEER CONSENT FORM
Student’s Name : Rachael Lalmalsawmkimi
Guides Name : Dr. Radhika Krishnaswamy
Msc. Mlt ( Biochemistry)
Associate Professor
Dept. of Biochemistry.
Title : Evaluation of Serum Albumin in individuals having Hyperlipidemia
Investigation : Serum Albumin
This study has been explained to me and I understand –
a) What the study involves
b) Refusal to participate will not affect my treatment in any cause.
c) That I may withdraw at any time
I therefore agree to take part in the study.
Signature of Subject: ______________________
Full Name
: _______________________
Date
: _______________________
Phone Number / Address: ___________________