A Comparative Study of Baseline Heart Rate Variability, Sleep Quality, and Oxidative Stress Levels in Hypertensive and Normotensive Subjects: A Cross-Sectional Study

Malondialdehyde estimate

Sample collection

Three milliliters of venous blood was drawn from each subject in a sterilized fashion from the median cubital vein into a plain red bottle (BD, USA). Whole blood was allowed to clot for 10 minutes before being centrifuged at 3000 rpm for five minutes to separate serum, which was then collected in a sterile Eppendorf microcentrifuge tube (Eppendorf, Germany) and stored at -800 ° C until next procedure .

MDA Analysis

Instruments used:

20 ml graduated tubes

Container filled with ice


Boiling water bath


ELISA plate

ELISA reader

Reagents used and their preparation

MDA: MDA is needed to prepare different standard concentrations against which samples are quantified.

TCA: 20% TCA was prepared by weighing 20 g pure solute trichloroacetic acid into 100 ml DDW.

H2SO4: 0.05 M sulfuric acid was prepared by adding 2.8 ml of concentrated sulfuric acid to 1 liter of DDW.

Sodium sulfate solution: A 2 M sodium sulfate solution was prepared by dissolving 28.4 g of anhydrous sodium sulfate in 90 mL of DDW with constant heating and stirring. An additional final volume is increased to 100 ml with DDW.

TBA: 0.67% thiobarbituric acid was prepared by dissolving 670 mg of TBA in 100 mL of 2 M sodium sulfate solution.

n-Butanol/n-butyl alcohol: readily available commercial n-butyl alcohol was used.

Sterile/double distilled water

Standard preparation

MDA standard solution was prepared from the purchased 500 μM stock solution (EZAssay TBARS estimation kit, Himedia, India).

Basic working standard for MDA: prepared by diluting 0.2 mL of stock solution in 10 mL of 0.05 MH2SO4 to obtain 10 µM of working stock solution.

Different concentrations of standards were prepared by diluting the working stock according to the following table:

Principle of MDA

Lipoproteins were precipitated from the serum sample by adding 20% ​​TCA. Malondialdehyde present in the sample reacts with thiobarbituric acid in sodium sulfate to form a pink-colored complex. The intensity of the colored complex is directly dependent on the concentration of MDA levels present in the serum sample.

The developed colored complex was then extracted into n-butanol, which was finally read for absorbance readings at 530 nm (green filter) using a filter colorimeter or ELISA reader. The method was standardized using various concentrations of standard solution and a standard curve was drawn on graph paper to measure the reading of the test results.

MDA estimation procedure

Three clean, dry and labeled test tubes were placed in the test tube rack, and the following items were carefully pipetted into the respective tubes as in the table seven


TEST (µl)


WHITE (µl)



Standard (10μM/L)


Double distilled water


20% TCA




The tubes were left to stand for 10 minutes at room temperature

The tubes were then centrifuged at 3500 rpm for 10 minutes.

The supernatant was decanted and the precipitate was treated with the following reagents.





0.67% TBD




All tubes were heated in a boiling water bath at 90ohC for 30 minutes.

The tubes were then chilled on ice.





With vigorous stirring the resulting chromogen was obtained.

Separation of the organic phase by centrifugation at 3000 rpm for 10 minutes was carried out

The organic phase was filtered and 200 μl pipetted respectively into different wells of the ELISA plate.

The ELISA plate wells were mixed well and read at 530 nm on the ELISA reader.

Patient case form

Participant number: Name:

Age/Sex: Religion

History reported by: Address:

Telephone number: Occupation

Presentation of complaints: (with duration in chronological order)


History: (with details of treatment and outcome status)

Treatment history: (including details of out-of-hospital treatments)

Family and close history:

Personal story:

1. Education-

2. Married/single –

3. Profession (monthly income, if any) –

4. Habits (details of smoking/alcohol) –

5. Details of hygiene habits (e.g. hand washing, sewage disposal, etc.) –

6. Menstrual history (if female) –

seven. Sexual history –

All STDs – (details including cure)

8. Sleep pattern and mental state –

9. A diet including recent detailed dietary history up to 2 days prior to onset of symptoms-

ten. Vaccination status –

11. Travel History –

Social background: (including socio-economic status, surrounding lifestyles, domicile and habitus, occupation)

Physical examination: (with consent and hand hygiene)

1. Mental state –

2. General Appearance –

3. Vitals: PR – , RR – , BP – , Temp – SpO2

4. Skin – Height: Weight: BMI:





5. Chest (respiratory system)

6. Chest (cardiovascular)

seven. Abdomen

8. Genitourinary-rectal examination –

9. Musculoskeletal examination –

ten. Neurological examination –

11. Survey planned (to be sent for all participants)-

Complete blood count

· Blood urea nitrogen

· Serum creatinine

· Serum electrolytes (Na, K, Cl, Ca)

· Pulmonary radiography

· 12-lead ECG

· Urine analysis

· Plasma blood glucose

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