European Journal of Cardiovascular Medicine

A common medical emergency is a stroke. The adoption of less healthful lifestyles is the reason behind the sharp increase in incidence in many developing nations. Treatment is still ineffective despite its difficulty. The best course of action is prevention, but it can be difficult to predict strokes, therefore a thorough analysis of risk factors is crucial. The sudden death of brain cells as a result of insufficient blood flow is known as a stroke or cerebral vascular accident. "The rapid development of clinical signs and symptoms of focal neurological disturbance lasting more than 24 hours or leading to death with no apparent cause other than vascular origin" is the clinical definition of stroke given by the World Health Organization.1.

After coronary heart disease, stroke is the second most prevalent cause of death globally2 (CHD). According to a study on the global burden of disease, India's population-based yearly stroke incidence was estimated to be 89 per 100,000 in 20053. This number is expected to rise to 91 per 100,000 in 2015 and 98 per 100,000 in 2030. It is one of India's biggest health issues. Both communicable and non-communicable diseases are a double burden for developing nations like India. The range of the estimated adjusted prevalence rate of stroke in rural regions is 84 to 262/100,000, whereas in urban areas it is 334 to 424/100,0004. According to current demographic surveys, the incidence rate ranges from 119 to 145/100,0005. Research indicates that 9.2–30% of admissions to neurological wards and 0.9–4.5% of all medical admissions are related to stroke6. Research has indicated a rise in the incidence of stroke among younger adults in India (less than 40 years old), accounting for 10 to 15% of all cases.7 80% of stroke cases worldwide are expected to occur in low- and middle-income nations by 2054, primarily in China and India, according to WHO estimates.

The study was conducted on 100 patients of cerebrovascular accident admitted in King George Hospital, Andhra Medical College, Visakhapatnam. in both male and female ward & intensive medical care unit between October 2022 to September 2023.

COLLECTION OF DATA

• Sample size: 100 patients
• Case control study with 50 patients having ischemic stroke and 50 patients having haemorrhagic stroke. The patient in both the groups did not have cardiac diseases .
• Patients were interviewed for demographic data such as age and sex, history of other comorbid conditions along with presenting complaints were noted. Further these patients were subjected to a physical examination for detailed neurological clinical examination and along with appropriate investigations. These findings were recorded on a predesigned and pretested proforma.

INCLUSION CRITERIA:

• Age:35-70 years
• Both Male and Female
• Patient admitted with CVA with neurological weakness

EXCLUSION CRITERIA:

• Patient refusal.
• Pre-existing cardiac diseases
• Presumptive diagnosis of stroke with no evidence on CT
• CVA with tumour
• CVA with trauma
• Liver disease

SAMPLE COLLECTION

Under aseptic precautions, fasting venous blood sample of 6ml was collected from each subject .2 ml of collected blood was transferred to a plain tubes ,and 2ml of blood to a EDTA containing vacutainers. The vacutainers containing the blood samples were kept at room temperature for 30 min and were centrifuged at 2000 g for 15 minutes for clear separation of serum. The following parameters were estimated, immediately after the serum was separated.

ESTIMATION OF HDL CHOLESTEROL:

METHOD: Phosphotungstic acid method, endpoint

REAGENT COMPOSITION:

HDL cholesterol standard – 25mg/dl

SAMPLE: Unhemolysed serum used PRECIPITATION:

Precipitation of LDL, VLDL and Chylomicrons done as follows:

Mixed well and the reaction mixture was allowed to stand for 10 min at room temperature, centrifuged at 4000 rpm for 10min and obtain a clear supernatant. The supernatant was used to determine the concentration of HDL cholesterol in the sample.

ASSAY PROCEDURE:

Mixed well and incubated for 10 min at room temperature.

The absorbance of the standard and the test samples were read at 505 nm against reagent blank.

CALCULATION

HDL cholesterol (mg/dl) = Absorbance of test x conc.of standard x dilution factor

Absorbance of standard

=  Absorbance  of  the  test x 25x3 Absorbance of the standard

= Absorbance of the test  x 75 Absorbance of the standard

Linearity-upto 125mg/d

ESTIMATION OF TOTAL CHOLESTEROL:

METHOD: Cholesterol Oxidase-Peroxidase Enzymatic, endpoint method.

PRINCIPLE:

The free cholesterol, liberated from the cholesterol esters by cholesterol esterase, is oxidised by cholesterol oxidase to cholestenone with the simultaneous production of hydrogen peroxide. The hydrogen peroxide reacts with 4 amino antipyrine and a phenolic compound in the presence of peroxidase to yield a red coloured complex.

1. Cholesterol ester +water CHE cholesterol + fatty acid

2. Cholesterol + oxygen CHO cholest-4-en-3one +H 2O 2
3. 2 H2O2 + 4AAP+ phenol POD quinoneimine dye + 4H2O

• CHE- Cholesterol esterase • CHO- Cholesterol oxidase

• 4AAP- 4 amino antipyrine • POD- Peroxidase

Absorbance of quinoneimine formed is directly proportional to cholesterol concentration.

REAGENT:        Reagent-1(Enzyme/chromogen)

Reagent-1A(BUFFER) Cholesterol standard- 200mg/dl

Reconstituted reagent:

Dissolve the contents of one bottle of the reagent-1 with one bottle of reagent-1A.

Mixed well and incubated for 10 min at room temperature. The absorbance of the test and standard were read against reagent blank at wavelength of 505 nm .CALCULATION:

Cholesterol(mg/dl) =   Absorbance  of  test  ×concentration  of

standard(mg/dl) Absorbance of standard

REFERENCE RANGE: 150-200 mg/dl

Linearity –up to 750 mg/dl Sensitivity- 1mg/dl

INTERFERENCE: Hb upto 200mg/dl,Ascorbate upto 12mg/dl,Bilirubin upto 10mg/dl and Triglycerides upto700 mg/dl do not interfere with the test.

ESTIMATION OF TRIGLYCERIDES:

METHOD: GPO-PAP method, endpoint

METHODOLOGY:

Colorimetric, enzymatic method with glycerol phosphate oxidase. PRINCIPLE:

LPL

Triglycerides + H2O          Glycerol + free fatty acids GK

Glycerol + ATP   Glycerol 3 phosphate + ADP GPO

Glycerol 3 phosphate + O2              DAP + H2O2

PEROXIDASE

H2O2 + 4AAP+3,5-DHBS               Quinoneimine dye + 2H2O LPL- Lipoprotein

lipase     GK- Glycerol kinase

GPO- Glycerol Phosphate Oxidase DAP-Dihydroxy Acetone Phosphate

ATP- Adenosine Tri Phosphate       4AAP- 4Amino Anti Pyrine

DHBS-3,5Dichloro-2Hydroxy Benzene Sulfonate

Lipoprotein lipase catalysed hydrolysis of triacylglycerol, yield glycerol which is phosphorylated by glycerol kinase using ATP to glycerol-3- phosphate which upon oxidation yields di hydroxy acetone phosphate and hydrogen peroxide. The hydrogen peroxide reacts with phenolic compound and 4amino antipyrine to form a coloured complex.

The intensity of Quinoneimine dye formed is proportional to the triglyceride concentration in the sample when measured at 505 nm (500- 540nm).

REAGENT:

Reagent 1(Enzymes/chromogen) Reagent 2(Buffer)

Triglycerides standard concentration- 200mg/dl

REAGENT PREPARATION:

The working reagent was prepared by mixing 4 parts of R1 with 1 part of R2. Stable for 90 days at 2-8 ◦C.

Sample: Unhemolysed serum collected after 12 hrs of fasting.

ASSAY PROCEDURE:

Mixed and incubated for 10min, at room temperature . Absorbance was read at 505nm for standard and sample against reagent blank.

CALCULATION:

Triglycerides(mg/dl)          = Absorbance of test          x             Concentration      of standard Absorbance of standard.

REFERENCE VALUES:

VLDL is estimated by Triglycerides/5.LDL

TABLE: 1 Comparison of Mean HDL between Ischemic and Hemorrhagic stroke

This table shows Mean HDL of ischemic stroke which is lower than Hemorrhagic stroke ,whose P value is 0.000 which is statistically significant.

Table: 2 comparison of mean lipid              profile between  ischemic              and Hemorrhagic stroke

This table shows Mean Total Cholesterol,Triglycerides,VLDL and LDL whose values are higher in ischemic stroke than Hemorrhagic Stroke.

Table: 3 Comparison of Mean BP between Ischemic and Hemorrhagic stroke

This table shows mean systolic and diastolic BP of Ischemic stroke and Hemorrhagic stroke . The Mean Systolic and Diastolic BP of Hemorrhagic stroke is higher than Ischemic stroke and the P value is

0.000 which is statistically significant.

Table: 4 Comparison                      of           Mean     Age        &RBS    between Ischaemic and Hemorrhagic stroke

(i)This table shows Mean age of Hemorrhagic stroke which is statistically significant (P Value 0.030 )than Mean Ischemic Stroke.(ii) This table also shows Mean RBS of ischemic stroke is more than Mean RBS of Hemorrhagic Stroke.

Table: 5 PERCENTAGE OF SITE OF LESION IN HEMORRHAGIC STROKE

Table: 6 PERCENTAGE OF ARTERY INVOLVEMENT IN ISCHEMIC STROKE

The present study done with 100 subjects (50 were ischemic stroke and 50 were Haemorrhagic stroke) supports the fact that measurement of Lipid profile was useful in predicting the risk factors for stroke.

The Mean value of HDL of Haemorrhagic stroke (39.36) was higher when compared to the Mean Ischemic Stroke (32.36), which is statistically significant with p value <0.05.

The mean values of Total Cholesterol (161.22), Triglycerides (159.56), VLDL(31.90) and LDL (96.96) of ischemic stroke were higher than Haemorrhagic Stroke, whose mean values of Total Cholesterol (159.56),Triglycerides (157.14),VLDL (31.46 )and LDL( 88.88).

The mean Systolic (161.28) and Diastolic BP (97.84) of Haemorrhagic stroke was higher than Ischemic stroke mean Systolic BP (135.60) and Diastolic BP (81.48) and the P value was <0.05, which is statistically significant

The mean age of Haemorrhagic stroke (58.08 ) was higher than mean ischemic Stroke (53.02) and the P value was <0.05, which is statistically significant.

The mean random blood sugar of ischemic stroke (150.58) was higher than Haemorrhagic Stroke (133.72).

The percentage of site of lesions in haemorrhagic stroke were putamen-34%, thalamus-18%, multiplesite-16%, Temporoparietal-14%, Brainstem-8%, Frontoparietal-6% and cerebellum-4%.

The percentage of artery involvement in ischemic stroke were Middle cerebral Artery-48%, Anterior CerebralArtery-14%, Vertebro basilar Artery-18% Internal Carotid Artery- 12%, and Multiple sites- 8%.

• This study shows HDL was significantly decreased in ischemic stroke than hemorrhagic stroke. whereas HDL was high in hemorraghic stroke.
• Total cholesterol, Triglycerides, LDL, VLDL were more in ischemic stroke than
• hemorrhagic stroke.
• Random Blood sugar was more in ischemic stroke than hemorrhagic stroke.
• Blood pressure was more in hemorrhagic stroke than ischemic stroke.
• Hemorrhagic stroke patients were older age group than ischemic stroke .
• The order of site of lesions in hemorrhagic stroke were putamen> thalamus> multiple               site>Temporo
• parietal>Brain stem>Frontoparietal>cerebellum.

The order of artery involvement in ischemic stroke were Middle cerebral Artery >AnteriorCerebralArtery >Vertebrobasilar Artery>Internal Carotid Artery > Multiple sites

This study shows that there is a significant decrease in HDL in ischemic stroke than Hemorrhagic stroke. As the Reverse Cholesterol Transport, Anti-inflammatory activity, Anti oxidative activity, Anti apoptotic activity, Endothelial repair, Anti thrombotic activity, Anti infectious activity of HDL are reduced due to decreased HDL leads to ischemic stroke than Hemorrhagic stroke.

Based on the results obtained from the present study HDL is used as an early predictor of atherosclerosis and ischemic stroke. By measuring the HDL earlier, early intervention measures by pharmaceutical means or by dietary means can be done to increase the HDL level to decrease the morbidity and mortality of stroke.

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