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Materials and Methods
The study was conducted at School of Health Sciences, Kannur
University Thalassery Campus, Palayad, and Pushpagiri Institute of Medical
Sciences and Research Center, Thiruvalla, Kerala, India. One hundred and
twelve clinically proved patients below the age of 65 years formed the test
group. Fifty age and sex matched control subjects from among the siblings of
the patients and staffs of the institute formed the control groups. None of the
subjects were suffering from autoimmune disease, and had no liver and renal
involvement.
Detailed
clinical,
epidemiological
and
anthropometric
characteristics were recorded using proforma (Appendix). Ten ml of fasting
venous blood was collected from all the subjects after getting the informed
consent. Three ml of blood was immediately transferred aseptically to
heparinized vacuutainers and used for invitro mutagen sensitivity analysis.
The remaining 7ml of blood was allowed to clot and serum
separated and the following investigations were carried out in the serum
sample.
1.
Total cholesterol
2.
High density lipoprotein cholesterol (HDL)
3.
Low density lipoprotein cholesterol (LDL)
4.
Triglycerides
5.
Malondialdehyde (MDA)
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6.
Ascorbic acid (Vitamin C)
7.
Glutathione (GSH)
1. Total Cholesterol (Rifai et al, 1999)
Cholesterol is estimated in serum by enzymatic direct quantitative
determination on Roche automated clinical chemistry analyzers. Cholesterol
esters are enzymatically hydrolyzed by cholesterol esterase to cholesterol and
free fatty acids. Free cholesterol, including that originally present, is then
oxidized by cholesterol oxidase to cholest-4-en-3-one and hydrogen
peroxide. The hydrogen peroxide combines with 4-amino phenazone phenol
to form a chromophore (quinoneimine dye) which may be quantitated at 500550 nm.
CHOL: ESTERASE
Cholesterol esters
Cholesterol + O2
Cholesterol+ Fatty acids + H2O
CHOL: OXIDASE
2H2O2 + 4-AminoPhenazone
+4-Chlorophenol
PEROXIDASE
Cholest-4-en-3-one + H2O2
4(p-benzoquinone-monoimino)
-phenazone +HCl+ 4 H2O
2. HDL Cholesterol (Sugiuchi et al, 1995)
HDL Cholesterol is estimated in serum by homogenous enzymatic
colorimetric assay on Roche automated clinical chemistry analyzers. In the
presence of magnesium sulfate, dextran sulfate selectively forms watersoluble complexes with LDL, VLDL and chylomicrons which are resistant to
PEG-modified enzymes. The cholesterol concentration of HDL-cholesterol is
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determined enzymatically by cholesterol esterase and cholesterol oxidase
coupled with PEG to the amino groups (approx. 40%). Cholesterol esters are
broken down quantitatively into free cholesterol and fatty acids by
cholesterol esterase. In the presence of oxygen, cholesterol is oxidized by
cholesterol oxidase to Δ4 -cholestenone and hydrogen peroxide. In the
presence of peroxidase, the hydrogen peroxide generated reacts with 4amino-antipyrine and HSDA to form a purple-blue dye. The color intensity
of this dye is directly proportional to the cholesterol concentration and is
measured photometrically.
Homogenous Enzymatic colorimetric assay.
PEG - CHOL ESTERASE
HDL Cholesterol esters
+ H2O
HDL Cholesterol + O2
H2O2 + 4-AAP
+HSDA + H2O+ H+
HDL Cholesterol + Fatty acids
PEG - CHOL: OXIDASE
PEROXIDASE
Δ4 -Cholestenone + H2O2
Purple blue pigment (585 nm)
+ 5 H2 O
HSDA - Sodium N-(2-hydroxy-3-sulfoprpyl)-3,5-dimethoxyaniline
4-4- AAP -4- Amino Anti Pyrine
3. LDL Cholesterol (Armstrong and Seidel, 1985)
LDL Cholesterol is estimated in serum by homogenous enzymatic
colorimetric assay on Roche automated clinical chemistry analyzers. Direct
determination of LDL-cholesterol takes advantage of the selective micellary
solubilization of LDL-cholesterol by nonionic detergent. Cholesterol esters
are broken down quantitatively into free cholesterol and fatty acids by
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cholesterol esterase. In the presence of oxygen, LDL cholesterol is oxidized
by cholesterol oxidase to Δ4-cholestenone and hydrogen peroxide. In the
presence of peroxidase, the hydrogen peroxide generated reacts with 4aminoantipyrine and HSDA to form a purple-blue dye. The color intensity of
this dye is directly proportional to the cholesterol concentration and is
measured photometrically.
Homogenous Enzymatic colorimetric assay.
DETERGENT
LDL Cholesterol esters
Fatty acids
+ H2O
Cholesterol + Free
CHOL: ESTERASE
CHOL: OXIDASE
Δ4 Cholestenone + H2O2
LDL Cholesterol + O2
PEROXIDASE
2 H2O2 + 4-AAP
+HSDA + H2O+ H+
Purple blue pigment (585 nm)
+ 5 H2 O
HSDA -Sodium N-(2-hydroxy-3-sulfoprpyl)-3,5-dimethoxyaniline
4-AAP - 4- Amino Anti Pyrine
4. Triglycerides (Mc Gowan et al, 1983)
Triglycerides in the serum were estimated by enzymatic GPO-PAP
method on Roche automated clinical chemistry analyzers. The triglycerides
in the sample are hydrolysed by a combination of microbial lipases to give
glycerol and fatty acids. The glycerol is phosphorylated by ATP in the
presence of glycerol kinase (GK) to produce glycerol-3-phosphate. The
glycerol-3-phosphate is oxidised by molecular oxygen in the presence of
GPO (glycerol phosphate oxidase) to produce hydrogen peroxide and
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Dihydroxyacetone phosphate (DHAP). The hydrogen peroxide produced
then reacts with
4-aminophenazone and 4-chlorophenol, catalysed by
peroxidase (POD) to give a red dye with an absorbance maximum at 500 nm.
The increase in absorbance is proportional to the triglyceride content of the
sample.
LIPASE
Triglycerides + 3 H2O
Glycerol + 3 Fatty acids
GK, Mg2+
Glycerol + ATP
Glycerol-3-phosphate
O2
Glycerol-3-phosphate + ADP
GPD
2H2O2 + 4-AminoPhenazone
+4-Chlorophenol
Dihydroxyacetone phosphate +
+ H2O2
POD
4(p-benzoquinone-monoimino)
-phenazone +HCl+ 4 H2O
ll the reagents, calibrators, controls used for Serum Triglyceride,
Serum Total Cholesterol, Serum HDL Cholesterol and Serum LDL
Cholesterol were from Roch Diagnostics and the analysis was carried out in
fully automated analyzer, Hitachi 912. Quality control was done with
Precinom Universal, Precipath Universal, Precinorm lipids and Precipath
lipids of Roch Diagnostics.
5. Malondialdehyde [MDA]
The assay is based on the reaction of malondialdehyde (MDA) with
thiobarbituric acid (TBA); forming a MDA-TBA2 adduct according to a
modified version of Satoh’s (1978) and Yagi’s (1984) methods.
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In brief, 0.5 ml serum was mixed with 4 ml 1/12 N H2SO4 in a
centrifuge tube and shaken gently. After 0.5 ml 10% phosphotungstic acid
was added to the tube and it was left to stand at room temperature for 5
minutes, the mixture was centrifuged at 3000 rpm for 10 minutes. The
supernatant was discarded and the sediment was mixed with 2.5 ml 1/12 N
H2SO4. The centrifugation was repeated and the supernatant was discarded
again. The sediment was resuspended in 2.5 ml 1/12 N H2SO4 and in fresh 3
ml 0.2% thiobarbituric acid reagent, mixed thoroughly and heated in a bath
of boiling water for one hour. After cooling in cold water, the resulting
chromogen was extracted with 3.0 ml n-butyl alcohol by vigorous shaking.
The organic phase was separated by centrifugation at 3000 rpm for 10
minutes, and its absorbance was recorded at a wavelength of 532 nm using
UV-VIS Spectrophotometer. The level of absorbance was converted into
nmol/ml MDA from a standard curve generated with 1, 1, 3, 3- tetraethoxypropane (SIGMA).
6. Ascorbic Acid [Vitamin C] (Varley, 2002)
Protein free supernatant was prepared by adding 2.0 ml of serum and
6.0ml of 5% TCA. To 0.5 ml protein free supernatant 1.5 ml DTC reagent (2,
4- dinitrophenyl hydrazine-thiourea-CuSO reagent) was added. 1.5 ml DTC
reagent and 0.5ml distilled water was taken as blank. All the tubes are
incubated at room temperature for1 hour. Then 2 ml ice cold sulphuric acid
was added to the tubes. The intensity of the yellow colour developed was
read at 520 nm in UV VIS Spectrophotometer. A standard graph was plotted
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with standard concentration adjusted to 0.25 mg%, 0.5 mg%, 1 mg%, 1.5
mg%, and 2 mg%.
7. Glutathione [GSH] (Beutler and Kelley, 1963)
5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB) and glutathione (GSH)
react to generate a yellow coloured product 2-nitro-5-thiobenzoic acid and
glutathione disulfide (GSSG). Protein free supernatant was prepared by
adding 3.8 ml precipitating reagent and added 200 µl of serum sample and
1ml distilled water in a test tube. Mixed well and filtered through a coarse
filter paper in to a centrifuge tube. Centrifuged and collected the supernatant.
To 1 ml filtrate added 0.5 ml DTNB reagent and 4 ml phosphate buffer as
test. A blank was taken with 0.5 ml DTNB reagent, 4 ml phosphate buffer,
0.6 ml distilled water and 0.4 ml precipitating reagent. A standard tube was
taken by adding 1 ml GSH standard (20 mg %), 0.5 ml DTNB reagent and 4
ml phosphate buffer. All the tubes were capped and mixed by inversion and
read the OD at 412 nm within four minutes in UV VIS Spectrophotometer.
8. Invitro Mutagen Sensitivity Analysis
Peripheral blood was collected aseptically by venipuncture.
Peripheral blood lymphocyte micro culture was performed as described by
Moorhead et al (1960) for determining the mutagen sensitivity. The culture
medium was RPMI 1640, supplemented with 20% fetal calf serum, 0.5ml
phytohaemagglutinin, 100-units/ml penicillin and 100 µg/ml streptomycin.
Bleomycin was added to induce chromosomal breakage according to the
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method of Hsu et al (1985). 0.03 units/ml of bleomycin treatment was given
5hrs (late S-G2 phase) before harvesting (66th hour).
The cultures were incubated for 72 hrs at 370C. At the end of 70th hr
all the cultures were treated with colcemid (0.04µgm/ml) to accumulate
mitoses before harvesting. The cells centrifuged at 1500 rpm for 10 minutes,
removed the supernatant. Re-suspended in 10 ml warm KCl hypotonic
solution (0.075M) after this added 2 drops of freshly prepared fixative
(Methanol: Acetic acid) in 3:1 ratio. The tube was centrifuged again at 1500
rpm for 10 minutes. This step was repeated many times until the supernatant
was clear and the cell button gets white.
Dropped the suspension drop by drop (7-8 drops) on pre cleaned
chilled slides with a fine bore pipette, flame the slide on spirit lamp, blow on
the material gently, dried and stored it in a slide box for ageing. The slides
were stained with Giemsa.
For mutagen induced chromosome sensitivity analysis the mean
number of breaks/cell (b/c) was calculated by the following formula. Only
frank chromatid breaks were scored.
Calculation
Number of breaks counted
X 46
Mean number of breaks/cell (b/c) = Total number of metaphase score
100
The frequency of breaks was expressed as b/c for comparison. Any
individual expression ≥0.8 b/c was considered sensitive and that ≥1.0 b/c was
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Geimsa Stained Metaphases
Metaphases showing chromatid breaks
considered hypersensitive. A minimum of 100 metaphases per culture was
scored and data were analyzed. The frequency of chromatid breaks was
considered as a measure of an individual’s DNA repair capacity.
All the chemicals and standards for MDA, ascorbic acid, GSH and
invitro mutagen sensitivity analysis were procured from Sigma.
Statistical Analysis
Results were expressed as mean ± SD. Independent sample ‘t’ test
was performed using SPSS (windows version 17.0) for comparing various
risk/lifestyle factors, biochemical and genetic characteristics such as total
cholesterol, LDL cholesterol, HDL cholesterol, MDA, ascorbic acid, GSH
and mean b/c value of the study subjects and the control subjects. Analysis of
variance (ANOVA) was done to compare the mean b/c value with and
without various risk/ life-style factors among subgroups. Probability values
of p < 0.05 were considered to be statistically significant. Association
between various risk/lifestyle factors, biochemical and genetic characteristics
and socio-economic variables and CAD is analyzed using chi-square test.
The contribution of various risk/lifestyle factors and biochemical and genetic
characteristics for coronary artery disease and the contribution of various
risk/lifestyle factors and biochemical characteristics for high mean b/c value
(sensitive) was studied by Logistic regression analysis.
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