Download 8. Garmendia JV et al . Nitric oxide in different types of hypertension

SYNOPSIS
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore
“STUDY OF LEVELS OF NITRIC OXIDE AND LIPID PEROXIDES IN
PREECALMPSIA”
Name of the candidate
:
Dr. Shobith Kumar Shetty
Guide
:
Dr. Hilda Priya D’Souza
Course and Subject
:
M.D. (Biochemistry)
Department of Biochemistry ,
A J Institute of Medical Sciences ,
Kuntikana, Mangalore – 575004.
2010
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1
Name of the candidate and
DR SHOBITH KUMAR SHETTY ,
address (in block letters)
POST GRADUATE RESIDENT,(MD)
DEPARTMENT OF BIOCHEMISTRY,
A J INSTITUTE OF MEDICAL SC IENCES
MANGALORE.
2
Name of the Institution
A J INSTITUTE OF MEDICAL SC IENCES
MANGALORE.
3
Course of study and Subject
MD BIOCHEMISTRY.
4
Date of admission to course
10-5- 2010
5
Title of the Topic : “STUDY OF LEVELS OF NITRIC OXIDE AND
LIPID PEROXIDES IN PREECLAMPSIA”
6
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR STUDY
Preeclampsia is considered to be one of the most significant health problems in human
pregnancy, complicating ~6–8% of all gestations over 20 weeks . This disease is one of
the leading causes of fetal growth retardation, infant morbidity and mortality, premature
birth and maternal death. Preeclampsia is defined as the triad of hypertension , proteinuria
and pathological oedema during pregnancy [1] .
Endothelial derived relaxing factor nitric oxide [NO] plays an important role in the
regulation of vascular tone, vascular permeability and blood coagulation, thus helping to
maintain circulatory hemostasis
[2].
Impairment of NO formation in the vessel wall will
lead to neutrophil activation and vasoconstriction in the maternal circulation and favour
platelet adhesion, aggregation and the consequent release of vasoconstrictor substances[3].
Nitric oxide is a highly reactive free radical with a very short half life , the labile nature of
nitric oxide makes it impossible to be analysed in serum and urine levels. It is rapidly
converted to nitrates and nitrites in the body; hence the concentration is parallel to nitric
oxide levels. Serum levels and urinary excretion of nitrates and nitrites reflect the total
production of NO in the body [4].
Significant correlation between preeclampsia & NO levels has been reported .Few studies
exploring nitrates & nitrite in blood samples of preeclampsia showed elevation[5][6],
decrease[7][8] and no change[9][10] with controls. Elevation of urinary nitrate & nitrite in
preeclampsia is studied. Increase level of plasma creatinine in patients with preeclampsia
indicating the impairment of renal function [11].
Oxidative stress is a component of preeclampsia, which could provide the linkage between
decreased placental perfusion and the maternal syndrome. Lipid peroxidation products,
primarily
measured
as
thiobarbituric
acid
reactive
substances
which
include
malondialdehyde (MDA) are increased in sera of women with preeclampsia. Decrease in
antioxidant status in contrary to lipid peroxides in preeclampsia
[12][13][14]
. The enhanced
generation of reactive oxygen species leads to decreased NO bioavailability [15].
In the present study, role of nitric oxide in preeclampsia & relationship between nitric
oxide & oxidative stress will be studied. Assessment of NO production in vivo by
measuring levels of nitrates & nitrites and creatinine in serum and urine in preeclamptic
patients & controls will be investigated. This relationship between preeclampsia and NO
may pave the way for novel therapeutic approaches such as nitric oxide in the management
of this difficult disease.
6.2 REVIEW OF LITERATURE
In normal pregnancy, profound physiological changes occur in the maternal
cardiovascular system, including in blood flow through uterine blood vessels, altered
responses to vasopressor agents & reduced peripheral resistance & blood pressure .These
cardiovascular changes ensure the adequate delivery of oxygen & nutrients to the fetus [16].
Human pregnancy is associated with a profound reduction in vascular resistance, evident
as early as the third week after conception
[17]
. Normal pregnancy is associated with
profound changes in maternal cardiovascular system which adopts to accommodate the
demands of the growing fetus. Blood volume and cardiac output increase by 40-50%
during the first trimester, despite this there is fall in the BP which is mediated by decrease
in total peripheral resistance. Multiple studies show that decrease in total peripheral
resistance is due to decreased response to vasospasmogenic agonists and increased action
of endothelium derived relaxing factor such as NO [2].
Preeclampsia is a multisystem disorder of pregnancy with clinical diagnostic features of
hypertension and proteinuria. Preeclampsia is associated with increased vascular reactivity
and vasoconstriction. The vascular, pathological features of preeclampsia have been
attributed to activation and injury of the endothelial cells
[18]
. In preeclampsia there is
increased systemic vascular resistance and decreased plasma volume and the reduced
action of EDRF may develop endothelial cell activation and establish preeclampsia [19].
Nitric Oxide is a biological mediator synthesized from L- Arginine by a family of nitric
oxide synthases. Nitric oxide is produced in many different cells and is involved in
regulation of physiological and pathological processes, such as inflammation and
metabolism
[20]
. Depending on cell types of nitric oxide produced in the enzymatic
reaction catalyzed by one of the three isoforms of nitric oxide synthase (NOS): Neuronal
NOS, Endothelial NOS and Inducible NOS [21].
Endothelial derived nitric oxide plays a role in the regulation of vascular resistance during
normal pregnancy and preeclampsia
[22][23]
. It has been postulated that endothelial
dysfunction and loss of endothelial mediated vasodilatation may account for increase
vascular resistance in women with preeclampsia [24].
Nitric oxide is a potent relaxant of vascular smooth muscle suggesting decreased nitric
oxide production may have adverse effect on placental haemodynamic function in
preeclampsia [25].
The numerous studies addressed to examine NOX production by measuring the NO
degradation products like nitrates & nitrites in both normal pregnancy & preeclampsia is
controversial thus an elevation
[5][6]
and decrease
[7][8]
or no change [9][10]] has been shown
in nitric oxide levels [NOX].
A study showed that urine concentrations of nitrate and nitrite normalized by creatinine
excretion were significantly decreased in preeclampsia compared to normal pregnant
women and this is due to reduced nitric oxide production by endothelial cells [11].
Oxidative stress is the principal causative factor, is reflected by increase in
Malondialdehyde and decrease in Total antioxidant activity. Impaired antioxidant activity
and the reduction of antioxidants could be the possible cause for the increased lipid
peroxidation observed which may cause damage to vascular endothelium resulting in
clinical symptoms of preeclampsia [12]. Oxidative stress is due to increased mitochondrial
activity and also because of placenta rich in macrophages and reactive chlorine species,
also might cause oxidative stress. It can provide a linkage between decreased placental
perfusion and maternal syndrome
[26][27]
. In preecalmptic patients antioxidants may be
used to greater extent to counteract free radical mediated cellular changes , resulting in the
reduction of plasma antioxidant levels[13][28][29].

OBJECTIVE OF THE STUDY
1) To compare the levels of serum and urinary nitric oxide in
subjects with preeclampsia with age & sex matched controls.
2) To compare the levels of serum and urinary lipid peroxides in
subjects with preeclampsia and age & sex matched controls.
3) To compare the total antioxidant in preeclamptic cases and age
and sex matched controls.
4) To examine the relationship betwe en the results in serum nitric
oxide/creatinine ratio & urine nitric oxide /creatinine ratio in
study and control group.
5) To determine the suitabilit y of serum & urine for estimating nitric
oxide as the potential biochemical marker of preeclampsia .
7.
MATERIAL AND METHODS:
7.1 SOURCE OF DATA:
The study comprises of 34 normal healthy pregnant subj ects & 34 cases of
preeclampsia aged between 20 to 35 years attending the Dept of Obstretics
& Gynecology AJ IMS Mangalore from the year 2010 -2012.
The diagnosis of preeclampsia was based on the definition of American
college of Obstetrics & gynecologists [ 3 0 ] .
1. Systolic blood pressure greater than 140mm Hg or a rise of aleast 30
mm Hg or
2. Diastolic blood pressure greater than 90 mm Hg or a rise of at le ast
15 mm Hg ( manifested on two occasions atleast 6 hours apart) and
3. Proteinuria of 300 mg or greater in 24 hours urine collection or
protein concentration of 1 gm/ L ( on two occas ions of atleast 6
hours apart)
INCLUSION CRITERIA
34 Primigravida with d iagnosed preeclampsia according to A.C.O.G with a n
age between 20 to 35 years in thir d trimester and 34 normotensive
primigravida women with no protienuria from third trimester and without
any age systemic or endocrine disorders and age matched with cases .
EXCLUSION CRITERIA :
Subjects with the history of diabetes, kidney disease, infections, severe anaemia (Hb<6
gm%), history of smoking , history of high blood pressure prior to pregnancy ,
multigravida & age above 35 yrs will be excluded.
7.2 METHOD OF COLLECTION OF DATA
7.2 a METHOD OF SAMPLE COLLECTION :
5ml of fasting blood sample will be collected aseptically from the anticubital vein using
plain vacutainers from 34 subjects each with and without preeclampsia. Serum will be
separated and analysed for the levels of serum nitric oxide , serum MDA, and serum Total
antioxidant status & serum creatinine.
5ml of urine will be collected in a clean , dry & sterile container for estimation of urinary
nitric oxide , MDA and creatinine .
7.2 b SAMPLE SIZE AND SAMPLING TECHNIQUE : A sample size of 68
consisting of 34 subjects each with and without preeclampsia will be selected by
purposive sampling technique.
7.2 c PARAMETERS TO BE STUDIED :




Serum nitric oxide and urinary nitric oxide by Greiss reagent method using
spectrophotometer [31] .
Serum and urine MDA by Thiobarbituric acid assay using spectrophotometer [32].
Serum total antioxidant status by using Phosphomolybdenum using
spectrophotometer [33].
Serum creatinine and urinary creatinine by Modified Jaffe’s using autoanalyser[34].
7.2 d TYPE OF STUDY: Case control study
7.2 e STATISTICAL ANALYSIS : collected data will be analysed by Unpaired `t’test,
7.3 Does the study require any investigations or interventions to be
conducted on patients or other humans or animals? If so, please describe
briefl y.
Yes, blood and urine samples are collected with aseptic precaution
after obtaining informed consent from patients.
7.4 Approval from ethical committee: Yes .
8
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