Lipid Concentration And The Extent Of Their Peroxidation In Nigerian Hypertensives
P Igbinaduwa, B Igbinaduwa, I Oforofuo
Keywords
blood pressure, cholesterol, hypertension, lipid, peroxidation, phospholipid
Citation
P Igbinaduwa, B Igbinaduwa, I Oforofuo. Lipid Concentration And The Extent Of Their Peroxidation In Nigerian Hypertensives. The Internet Journal of Health. 2007 Volume 8 Number 1.
Abstract
Plasma total cholesterol, triglycerides, high density lipoprotein cholesterol, low density lipoprotein cholesterol, Phospholipids and molonydialdehyde were determined in normotensives, hypertensives on anti – hypertensive drugs and hypertensives not on any anti hypertensive drugs aged between 30 – 60 years. Plasma samples were used.
Result were statistically significant in all parameters studied at the level of (p < 0.001) in normotensives, hypertensives on drugs and hypertensives not on drugs except in phospholipids where there was no statistical difference. Relationship between blood pressure and lipid concentration were established.
The product of lipid peroxidation, malonydialdehyde was statistically significant in hypertensives on anti-hypertensive drugs and hypertensives not on any anti-hypertensive drugs when compared to normotensive control at (p<0.001) indicating free radical generation as a result of oxidative stress. These finding indicates that there are high lipid and lipoprotein concentration in hypertensives.
The Research work was done at The Federal Medical Centre Asaba and The University of Benin.
Introduction
Hypertension has emerged as a significant public health problem in most developing countries of the world 1 . Uncontrolled hypertension remains a major threat to global health especially in developing countries such as Nigeria, where cardiovascular diseases mainly resulting from hypertension are still responsible for significant rates of death and disability 2 .
The major risk factors for cardiovascular disorder is associated with plasma cholesterol concentration and fractions known as low density lipoprotein (LDL) cholesterol and very low density lipoprotein (VLDL) cholesterol, whereas the fraction known as high density lipoprotein (HDL) cholesterol appears to have a protective role 3 . Also peroxidation of these lipids by free radicals is believed to be one of the important causes of cell membrane destruction and could be implicated in the pathogenesis of cardiovascular disorder 45 .
This study is aimed at evaluating the lipids most commonly implicated in hypertensives; assessing the lipid levels in hypertensives and normotensives and to find out if there is any variation in the lipid concentrations of hypertensive patients compared to normotensive patients and also to investigate lipid peroxidation assessed by plasma malonydialdehyde level in hypertensive patients attending the cardiovascular clinic at Federal Medical Centre Asaba.
Materials And Methods
Subjects For The Study
130 subjects composed of 30 hypertensives not on any drug therapy, 50 hypertensives on drug therapy and 50 normotensives attending cardiology clinic at Federal Medical Centre Asaba were assessed. The normotensives, apparently healthy subjects who were not suffering from any known disease at the time of this study have their systolic / diastolic blood pressure below 130/80 mmHg. The hypertensives, were newly diagnosed and were not on any anti hypertensive drug have their systolic / diastolic blood pressure above 130/80 mmHg. The hypertensives on drugs also have blood pressure above 130/ 80mmHg and above.
Fasting blood were collected from the subject with a 10ml syringe after a 12 hour fast overnight prior to venepucture.
Preparation Of Plasma
The blood specimen collected into lithium heparin containers were centrifuged within one hour of collection for 10 minutes using a Gallenkamp centrifuge. The plasma was separated from the red cell using a Pasteur pipette into containers containing no anticoagulant and stored frozen prior to analysis.
Estimation Of Plasma Total Cholesterol
The enzymatic colorimetric method bt Tinder was used 6 .
Reagents
4-Aminoantipyrine 0.5 mM, Phenol 6mM, 3,5-dichlorophenol 0.2mM, Cholesterol esterase ≥ 500kµ/l, Cholesterol oxidase ≥ 300kµ/l, Peroxidase ≥ 1200kµ/l , Phosphate buffer (pH 6.7) 70mM.
Principle
Figure 1
Procedure
To 1000ul of reagent in tubes meant for samples, standard and blank, 10µl of samples, standard and distilled water were added respectively. The content was mixed and incubated for 5mins at 37 ° C. The absorbance of the samples and standard was measured against reagent blank at 546nm
Calculation
Figure 2
Estimation Of Hdl-Cholesterol
Reagents
HDL-cholesterol precipitant (Phosphotungstic 4.8%, Magnesium chloride 3M)
Principle
Low density lipoproteins (LDL and VLDL) and chylomicron fractions are precipitated quantitatively by the addition of acid phosphotungstic in the presence of magnesium ions. After centrifugation the cholesterol concentration in the HDL (high density lipoprotein) fraction, which remains in the supernatant is determined.
Procedure
Into centrifuged tubes, 500µl of sample and 100µl of HDL-cholesterol precipitant was added, mixed and allowed to stand for 10mins at room temperature .The tubes were centrifuged at 4,000rpm.100µl of supernatant were assayed for cholesterol estimation with the procedure given above for total cholesterol estimation.
Calculation
Figure 3
Estimation Of LDL-Cholesterol
Reagent
Polyvinyl sulphate 0.7g/l EDTA Na2 5.0mM Polyethylene glycol monomethyl ether 170g/l
Principle
LDL-cholesterol can be determined as the difference between total cholesterol and the cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the presence of polyethylene glycol monomethyl ether.
Procedure
To 0.1ml of precipitant solution in a centrifuge tube 0.2ml of sample was added. The mixture was then mixed and allowed to stand at room temperature. The mixture was then centrifuge at 2,000g for 15mins and the supernatant cholesterol concentration was determined using enzymatic colorimetric method.
Calculations
LDL-cholesterol (mg/dl) =Total cholesterol (mg/dl) – 1.5 x supernant cholesterol (mg/dl)
Estimation Of Triglyceride In Plasma
The enzymatic colorimetric method by McGowan was used 7 .
Reagent
Pipes buffer pH 6.8 50mM, 4-chloro-phenol 4.2 mM, Magnesium aspartate 40 mM, 4-aminophenzone 0.35mM, ATP 2mM, lipase ≥9000 µ/l, Glycerol-Kinase ≥ 500µ/l, Glycerol-3-phosphate oxidase≥ 200µ/l, peroxidase ≥ 500µ/l.
Principle
Figure 4
Procedure
To 100µl of reagent in tubes meant for samples, standard and blank, 10µl of samples, standard and distilled water was added respectively. The content were mixed and incubated for 5mins at 37ºc.The absorbance of the samples and standard was measured against reagent blank at 546nm.
Calculation
Figure 5
Determination Of Malonydialdehyde, A Product Of Lipid Peroxidation
Lipid peroxidation end product was determined according to a modification of Buege and Aust 8
Reagents
15% w/v Trichloroacetic acid
0.25 mol/l Hydrochloric acid
0.375 W/v Thiobarbituric acid
Principle
Malonydialdehyde (MDA), a product of Lipid peroxidation when heated with thiobarbituric acid under acid condition form a pink colored complex which has a maximum absorbance at 532nm.
Procedure
One volume of plasma was mixed thoroughly with two volumes of stock solution of 15% trichloroacetic acid, 0.375% thiobarbiburic acid and 0.25 mol/l hydrochloric acid. The mixture was heated for 30mins in a boiling water bath. After cooling, the precipitate was removed by centrifugation at 1000g for 10 minutes. The absorbance of the sample was determined at 535nm and the concentration was calculated using the extinction coefficient, 1.56 X 10 5 m 1 cm 1
Estimation Of Plasma Phospholipids
Reagent
1. Chromogenic reagent
Solution i) 16g Ammonium molybdate was dissolved in 120ml distilled water.
Solution ii) 80ml of solution 1 was added to 40ml conc HCl and 10ml of mercury. The solution was shaken constantly for 30 minutes and filtered.
Solution iii) 200ml conc H2SO4 was added to the remaining solution i above cooled and added to the filtered solution ii. The chromogenic solution was obtained by adding 45ml methanol, 5ml chloroform and 20 ml distilled water.
2. Phospholipid standard; L- α phosphatidylcholine (1mg/ml)
3. Isopropanol
Principle
This involves complexing intact phospholipids with mercury-ammonium molybdate chromogenic reagent 9 . The absorbance was measured spectrophotometrically at 710nm
Procedure
0.2ml plasma was added to 3.8ml isopropanol. The mixture was centrifuged for 10 minutes at 1000g. 0.5ml of supernatant was pipetted into centrifuged tubes and evaporated to dryness at 90 ° C.0.4ml chloroform and 0.1ml chromogenic solution were added after thorough mixing, the tubes were heated at 100 ° C for 1 minute. After cooling at room temperature, the tubes were mixed and 3ml chloroform was added. At the end of 15minutes, the tubes were centrifuged at 1000g for 5minutes and the absorbance of the supernatants read at 710nm. Isopropanol was used as blank.
Calculation
Figure 6
Statistical Analysis
All measures of central tendencies and dispersion were analysed using descriptive statistics in statistical packages for social sciences (SPSS) statistical package. The non-parametric Kruskal-Wallis test was used to test for the significant difference.
Results
The plasma lipid concentration and malonydialdehyde were determined from a total of 130 subjects made up of normotensives, hypertensives on anti hypertensive drugs and hypertensives not on any anti hypertensive drugs and they were aged between 30-60 years.
Figure 7
Figure 8
Figure 9
Figure 10
KEYS
BPS = Systolic Blood Pressure in mmHg
BPD = Diastolic Blood Pressure in mmHg
HDL = High Density Lipoprotein Cholesterol
LDL = Low Density Lipoprotein Cholesterol
HOD = Hypertensives on Drugs
HND = Hypertensive not on Drugs
n = Number of subjects investigated
Discussion
The result of the work has shown that there is some variation in plasma lipids in relation to sex. The total plasma cholesterol was significantly higher in females than in the males. There was a significant difference in the lipid concentration and malonydialdehyde concentration between normotensives, hypertensive on hypertensive drugs and those not on any anti-hypertensive drugs.
Total cholesterol, triglycerides and LDL cholesterol were significantly higher in hypertensives not on any drug therapy than normotensives (P< 0.001). There was a significant difference in blood pressures, total cholesterol, HDL-cholesterol and LDL-cholesterol in hypertensives on antihypertensive drugs when compared to hypertensives not on any antihypertensive drugs, but triglyceride concentration increased marginally in hypertensives on antihypertensive drug than hypertensive not on any anti-hypertensive drug. The reason may be attributed to the effect of antihypertensive drug on triglycerides.
There were high levels of total cholesterol, triglycerides and LDL-cholesterol and low levels of HDL-cholesterol were observed. There was no significant difference in the phospholipids concentration (P>0.05) of normotensives compared to hypertensives.
In our study, we observed an increase in malonydialdehyde, a product of lipid concentration among the normotensives, hypertensives on anti-hypertensives and hypertensives not on any anti hypertensive drug. However there was no significant difference observed in the malonydialdehyde concentration of hypertensives on anti-hypertensive drugs compared to those not on any anti-hypertensive drugs. This may be attributed to the fact that anti-hypertensive drugs may not have any effect on lipid peroxidation.
Conclusion
The results obtained from this study indicate that lipid concentration are increased in hypertensive patients and if not controlled could lead to other cardiovascular disorders or possibly death. Regular laboratory investigations of lipid profile should be investigated along with blood pressure in hypertensives since lipid and lipoproteins are important diagnostic and predictive value in coronary heart diseases.
Correspondence to
Patrick Igbinaduwa, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Benin, Benin City. Phone: +2348037422420 Email: pigbinaduwa@yahoo.com
