A Modified Method to Determine Lipids Peroxidation in patients with HPV16 Cervicitis
Ali M.A. Al-Kufaishi1, Lamia A. M. Al-Mashhedy2, Bushra Jaber Al-Rubaie3
1Al-Furat Al-Awsat Technical University College of Health and Medical Techniques,
Department of Medical Laboratory Techniques, Kufa, Iraq 31003
2University of Babylon, College of Science, Chemistry Department
3University of Babylon, College of Medicine, Gynecology Department
*Corresponding Author E-mail: Kuh.ali@atu.edu.iq, dr.lamia71@yahoo.com, bushra6347@yahoo.com
ABSTRACT:
HIGH LIGHTS: HPV16 and related to cervical cancer; Oxidative stress that associated with HPV16 or anther microorganism that infected cervical; A modified method of lipids peroxidation uses a good marker to assessment the redox status. Objective: Application of modified methods to assessment of lipids peroxidation in patients with chronic cervicitis. Design and Methods: A comparative study to the estimation of lipids peroxidation of the patients with HPV16 through divided the cases into three groups, healthy women (G1), chronic cervicitis patients without HPV16 (G2) and chronic cervicitis patients with HPV16 (G3). Results: The results show the high levels of lipids peroxidation in the patients (51.89) for cervical mucus and (22.59) for a serum with HPV 16, where (37.51) in cervical mucus and (19) for serum in the patients without HPV 16, compared with healthy (6.95) in serum and (25.54) in cervical mucus. Conclusion: HPV 16 consist of more than 50% from cervical cancer, but is alone not enough to develop cancer, there are supplement agents such as raise of reactive oxygen species and reduction of antioxidants. The higher levels of lipids peroxidation in the serum and mucus of the patients compared with control. So, lipids peroxidation is a good biomarker to evaluate the redox status in the patients compared with control.
KEYWORDS: lipids peroxidation, MDA, HPV16, cervical cancer.
INTRODUCTION:
Free radicals and oxidative stress:
Free radicals, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in cells as a part from their normal metabolic processes. But the levels of ROS and RNS influence by several causes such as inflammation and when exposure to oxidants sources as UV, air pollution, administration some of the medications.
The most common ROS and RNS that correlated with diseases are superoxide radical (O2•ˉ), hydroxyl radical (•OH), hydrogen peroxide (H2O2), nitric oxide (NO•) and peroxynitrite (ONOOˉ). The imbalance between ROS and RNS with the antioxidants capacity form oxidative stress that correlated with several diseases such as diabetes, rheumatoid arthritis, cancer and Alzheimer disease[1].
In vivo, free radicals have a harmful effect on cellular components such as proteins, DNA and oxidation of lipids. The lipids peroxidation products are unstable compounds that are decomposition rapidly into variety sub-products. malondialdehyde (MDA) is the one from the most known secondary products for lipids peroxidation and considers a good biomarker for cell membrane injury. Another product of lipids peroxidation that used to the assessment of lipids membrane peroxidation are conjugated dienes, isoprostanes, ethane and pentane gases11 and 4-Hydroxynonenal[2,3].
Lipids peroxidation and Malondialdehyde:
Lipids peroxidation is a process that involved free radicals mediated chain of reactions that begin by the oxidative deterioration of polyunsaturated lipids. Lipids are the most target components from the biological membranes. The propagation step of oxidation involved a number of toxic compounds such as aldehydes and endoperoxides[4].
MDA can be defined as aldehyde with low molecular weight and consist from three-carbon atoms. Its generate by different mechanisms depending on the peroxides that possessed α or β unsaturation to the peroxide groups that have the ability to produce MDA by cyclization process[5]. The production of MDA represented by (Figure 1). ROS and RNS react with carbon-carbon double bond of polyunsaturated fatty acid (I). The delocalization of double-bound weakness carbon-hydrogen bond and lead to abstract hydrogen by free radical (II) and to generate peroxyl radical (III). The propagation process began with the reaction of peroxyl radical with another fatty acid (IV)[6]. The lipids hydroperoxide is unstable and degrades to give MDA and 4-hydroxy-2-nonenal[7].
Fig. 1: Formation of MDA by lipids peroxidation.
Since 1960, there are several techniques have been developed to quantitative determine of MDA as spectrophotometry or fluorimetry, gas chromatography, high-performance liquid chromatography (HPLC) and immunological techniques[8,9]. In this research, we use a modified method to determine MDA in human specimens as serum and cervical mucus for the patients with HPV16, chronic cervicitis compared with healthy women.
Human papillomavirus:
Human papillomaviruses (HPV) are small nonenveloped viruses and its diameter a proximately 55nm, genetically consist from double-strand DNA and capsid proteins. HPV infected epithelial and endothelial cells of the human and specifically squamous epithelia and generate warts. There are more than 180 different HPV genotypes have been characterized into mucosal and cutaneous HPV, within these groups the individual viruses divided into high risk (HR-HPV) and low risk (LR-HPV) according their ability to malignant progression. In some times the LR-HPV that cause localized benign warts can be developed to malignant progression if not treated[10,11].
Only one strand from the DNA of HPV is an active transcript, The organization genome of HPV divided to three major regions: early region (E) that consist 50% from all HPV genome and responsible about encodes nonstructural proteins, noncoding or long-chain region and represented 10%, and late region (L) that encodes capsid proteins (L1 and L2) and represented 40% from all HPV genome[12].
HPV and Cervical Cancer:
CaCx is the most common gynaecological malignant around the world especially in the developed countries[13,14]. HPV16 and 18 genotyping responsible for about 90% of CaCx[15]. After infection by HR-HPV, E6 and E7 oncogenes expressed to produce oncoproteins E6 and E7. E6 and E7 are oncoproteins that binding and with tumour suppressor proteins that regulate cell cycle p53 and retinoblastoma protein (pRb) at respectively. The association between E6 and E7 with p53 and pRb lead to degradation via the ubiquitin-proteasome system (UPS) after binding with ubiquitin-protein by ligase enzyme[16,17] (Figure 2).
MATERIALS AND METHODS:
Study Design:
A comparative study for healthy, chronic cervicitis patients with positive HPV 16 genotyping, and patients without HPV.
Sample Collection:
HPV 16/18 genotyping detection by real-time PCR by taken specimens as tissues by cervical scraping and as Pap smear with mucus via gynecologists in the hospital Imam Sadiq and maternity hospital and children in Babil province, where dilution with phosphate buffer solution (PBS) (dilution factor equal four), in addition to serum for the same persons. The specimens (serum, cervical mucus, and tissues) collected depend on the criteria of chronic cervicitis such as age, number of pregnancy or abortion, infection delay, smoking, vaginal bleeding, lower abdominal pain and vaginal pH.
Fig. 2: Degredation of p53 and pRb by ubiquitin proteasome system (UPS).
Sample Size Calculation:
The sample size calculated by applying the following equation [18]:
Where Z refer to Z-score (equal 1.65), d is the absolute marginal error equal 10%, P is the population (number of women that have cervicitis and undergo to cervical screen equal 8%), therefore the number of samples equal to twenty for each group, but have been taken more than 20 for the chronic cervicitis without HPV 16/18 and healthy control (Table 1). The samples were collected for a period of six months.
Table 1: Groups classification.
|
Groups |
Description |
No. of the patients |
|
G1 |
Healthy women |
40 |
|
G2 |
Women with chronic cervicitis without HPV16 |
40 |
|
G3 |
Women with chronic HPV16 cervicitis |
20 |
Viral DNA extraction:
Depended on viral gene-spin DNA extraction kit, can be extracted DNA and prepared to real-time PCR.
Real-time PCR procedure:
Have been used Bosphore HPV genotyping kit for real-time PCR to detect HPV 16 genotype [19,20].
Lipids peroxidation Assay:
Principle:
A modified method by thiobarbituric acid – reactive kinds assay was used to evaluate the lipid peroxide formed using egg-yolk homogenates as lipid-rich media[21].
Reagents:
1. Egg- yolk (10%) in deionized water.
2. Ferrous sulphate 0.07 M in deionized water.
3. Acetic acid 20% (pH adjusted to 3.5 with NaOH).
4. TBA 0.8%, prepared in 1.1% sodium dodecyl sulphate.
5. TCA 20% in distilled water.
6. n – butanol.
Procedure:
Must be prepared three tubes sample, standard, and blank as following:
|
Reagents |
Control / µl |
Sample / µl |
Blank / µl |
|
Egg-yolk |
250 |
-------- |
-------- |
|
Sample |
-------- |
250 |
-------- |
|
D.D.W |
-------- |
-------- |
250 |
|
All tubes were mixed well and the volume was made up to 1ml by adding distilled water |
|||
|
Incubated all tubes for 30 min at 25ºC |
|||
|
Acetic Acid |
Acetic Acid |
Acetic Acid |
Acetic Acid |
|
TBA |
TBA |
TBA |
TBA |
|
TCA |
TCA |
TCA |
TCA |
|
All tubes were vortexed and heated in a boiling water bath for 60 min and then cooling |
|||
|
2500 |
2500 |
2500 |
n- butanol |
All tubes were centrifuged at 3000rpm for 10 min. The absorbance of the upper organic layer was measured at 532nm.
Calculation:
The percent of lipid peroxidation was calculated by using the equation as below.
Total lipid peroxidation %
A1 : Absorbance of samples.
A2: Absorbance of the fully oxidized control.
Statistical analysis:
The data were entered into SPSS program version 23 to get on the variables as mean, standard deviation (SD), standard error (S.E), confidence interval and “one-way ANOVA. A P-value of ≤ 0.05 was considered to be significant”.
RESULTS AND DISCUSSION:
The results that appear from the amplification plot of RT-PCR its contain on the HPV16 genotyping in the women that suffering from abdominal pain, bleeding after intercourse and excretion more amount from cervical mucus. The viral DNA extraction by specific protocol after taken biopsy or cervical tissues by cervical scraping.
Fig. 3: Amplification plots of real-time PCR.
Table 2: Lipids peroxidation percent of serum and cervical mucus.
|
Groups |
Lipids peroxidation% (Mean ± SD) |
S.E |
Confidence Interval 95% |
*P-Value |
||
|
Lower |
Upper |
|||||
|
Serum |
G1 |
6.95±2.81 |
0.44 |
3.13 |
12.50 |
0.000 |
|
0.000 |
||||||
|
G2 |
19.00±4.04 |
0.63 |
12.50 |
24.38 |
0.000 |
|
|
0.01 |
||||||
|
G3 |
22.59±5.91 |
1.32 |
13.13 |
35.00 |
0.000 |
|
|
0.01 |
||||||
|
Mucus |
G1 |
25.54±2.79 |
0.44 |
21.74 |
31.06 |
0.004 |
|
0.000 |
||||||
|
G2 |
37.51±4.01 |
0.63 |
31.06 |
42.86 |
0.004 |
|
|
0.001 |
||||||
|
G3 |
51.89±4.56 |
1.02 |
43.48 |
57.76 |
0.000 |
|
|
0.001 |
||||||
(*P-Value ≤ 0.05)
Fig. 4: Results of Lipids peroxidation % of G1, G2 and G3.
In spite of HPV16 from the common causes that lead to CaCx but it’s not enough alone to develop cervical neoplasia, because there are many cases can be eliminated from HPV by immunological response only. So, there are supplement factors encourage to change cells morphology that infected by HPV such as free radicals.
The results of following results illustrate the significant elevated for lipids peroxidation for G3 and G2 when compared with G1 in serum and cervical mucus. Also, the results of lipids peroxidation of cervical mucus are extremely elevated compared with the results of serum (Table 2) and (Figure 4).
Lipids peroxidation consider a good marker to detect the redox status of cells because the cellular lipids are the main target for free radicals. The free radical that generate due to innate immune attack from neutral killer cells and macrophages such as hypochlorite and superoxide for the cells that infected by HPV or another microorganism. The other causes that rise from free radicals are smoking, low levels of antioxidants and lifestyle.
Superoxide convert into hydroxyl radical, where the last radical is responsible about cellular lipids peroxidation. Therefore, the untreated cases with HPV16 who suffering from oxidative stress can progression the cervical infection to cervical cancer.
In this study, there is a modified method to determine the lipids peroxidation, where use egg-yolk as a source for lipids that oxidized by ferrous sulphate, then react with TBA and compared with a sample to calculate the total lipids peroxidation.
CONCLUSION:
In brief, the application of this modified methods to the estimation of lipids peroxidation gives a good indicator to assessment patients redox status that suffering from cervical diseases compared with healthy. Where the results of lipids peroxidation in patients with HPV16 are higher than infected with another microorganism.
CONFLICT OF INTEREST:
There is no conflict of interest with authors.
ETHICAL CLEARANCE:
This research got on the approval of the Scientific Committee of my University and Health Ministry of Iraq”.
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Received on 23.11.2019 Modified on 29.12.2019
Accepted on 31.01.2020 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(9):4087-4091.
DOI: 10.5958/0974-360X.2020.00722.2