|Year : 2016 | Volume
| Issue : 2 | Page : 71-76
Correlation of histopathological observations of appendicitis tissue with glutathione, lipid peroxidation, and lactate dehydrogenase: An investigational study
Elroy Saldanha1, Leo Francis Tauro1, Arnadi Ramachandrayya Shivashankara2, Ashwin Alva1, Joseph Alukal3, Manjeshwar Shrinath Baliga3
1 Department of General Surgery, Father Muller Medical College, Mangalore, Karnataka, India
2 Department of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India
3 Father Muller Research Centre, Mangalore, Karnataka, India
|Date of Web Publication||8-Jun-2016|
Leo Francis Tauro
Department of General Surgery, Father Muller Medical College, Kankanady, Mangalore - 575 002, Karnataka
Source of Support: None, Conflict of Interest: None
Objective: The present study was conducted to investigate the levels of antioxidant glutathione (GSH), lactate dehydrogenase (LDH), and lipid peroxidation (LPx) in the excised appendicitis tissue and investigate as to whether there is any correlation with the histopathological grading and clinical scoring. Materials and Methods: Appendix was collected from people requiring appendectomy (n = 30) and people with normal appendix but requiring gastrointestinal (GI) surgery for some other GI diseases and willing for complementary appendectomy (n = 4). The levels of GSH, LPx, and LDH were evaluated and statistically compared. In addition, statistical analysis is used to observe for correlation between these parameters with histological grading and Alvarado scores. Results: When compared to the normal appendix, in the tissues of appendicitis, the levels of LPx were high and that of LDH and GSH were significantly low. A negative correlation for GSH and positive correlation for LPx were observed. With respect to LDH, a negative correlation for tissue was observed with both histopathological and clinical grading. Multiple regression analysis for the biochemical endpoint in tissue showed that these parameters contribute 67.9 and 30.1% toward the histopathological and clinical grading, respectively. Conclusions: The results of the study indicate that the levels of LPx are increased and that of LDH and GSH are decreased in appendicitis and they are dependent on the pathological and clinical manifestation.
Keywords: Alvarado score, appendicitis, glutathione, lactate dehydrogenase, lipid peroxidation
|How to cite this article:|
Saldanha E, Tauro LF, Shivashankara AR, Alva A, Alukal J, Baliga MS. Correlation of histopathological observations of appendicitis tissue with glutathione, lipid peroxidation, and lactate dehydrogenase: An investigational study. Saudi Surg J 2016;4:71-6
|How to cite this URL:|
Saldanha E, Tauro LF, Shivashankara AR, Alva A, Alukal J, Baliga MS. Correlation of histopathological observations of appendicitis tissue with glutathione, lipid peroxidation, and lactate dehydrogenase: An investigational study. Saudi Surg J [serial online] 2016 [cited 2022 May 27];4:71-6. Available from: https://www.saudisurgj.org/text.asp?2016/4/2/71/183676
| Introduction|| |
Appendicitis, an acute inflammation of the appendix, is a very common gastrointestinal (GI) disease needing immediate surgical intervention.  Appendicitis has a lifetime risk of approximately 8.6% in males and 6.7% in females and when neglected progress from a simple inflammatory condition to abscess, ileus, peritonitis, and may also result in the death of the patient.  Reports indicate that early diagnosis and treatment have minimal risk of mortality while when neglected and undiagnosed increases the risk of death, especially when perforation has ensued.  The diagnosis of acute appendicitis is predominantly based on clinical findings, and the Alvarado scoring system developed by Alvarado in 1986 for the diagnosis of acute appendicitis in emergency situation is globally the most reliable and followed scale. The Alvarado scoring system is easy to apply because it relies purely on clinical history, examination, and a few simple laboratory tests. 
The cause of acute appendicitis is unknown but is best suggested to be a multifactorial luminal obstruction, and aspects such as dietary and familial factors have all been suggested to play cardinal role in the underlying pathogenesis. , Reports also suggest that generation of free radicals and triggering of inflammatory reactions are implicated in the etiopathogenesis of many clinical conditions, and this also extends to acute appendicitis. , Previous studies have shown that patients with acute appendicitis have low levels of antioxidants and a concomitant increase in the levels of oxidative stress and lipid peroxidation (LPx) in the blood, thereby substantiating the hypothesis that free radical generation and the resulting stress does indeed have a role to play in the underlying pathogenesis. ,,,
The hypothesis of our study was that in appendicitis tissue, the levels of cellular antioxidant glutathione (GSH) would be less and that of LPx and lactate dehydrogenase (LDH) will be more. Therefore, in this study, we proposed to assess the levels of GSH, LPx, and LDH, the important markers of oxidative stress and cell death, in the excised tissue of patients with acute appendicitis and compared with normal appendix excised from volunteers requiring surgery for early colon cancer and willing for complementary appendectomy. In addition, we also subjected the data obtained from biochemical assays with both histopathological and clinical grading (Alvarado score) to assess for existence of any correlation between the various parameters with histopathological and clinical grading.
| Materials and Methods|| |
The present study was a collaborative endeavor of the Department of Surgery and Department of Biochemistry of the Father Muller Medical College, Mangalore, Karnataka, India. The study was performed after the approval of the Institutional Ethical Committee from January 2012 to December 2012. The inclusion criteria included patients with typical features of acute appendicitis (in accordance to the Alvarado score) and admitted for appendectomy, people with normal appendix but requiring GI surgery for some other GI diseases, and willing for complementary appendectomy. A written consent for the study was taken from all adult volunteers and patients while it was taken from one of the parents of children and adolescents below the age of 18.
The clinical scoring was carried out according to the Alvarado scoring system in the patients suspected to have acute appendicitis.  For correlation purpose, we used the mean score derived from adding the individual scores and dividing it by 10. The blood samples were collected preoperatively from the appendicitis patients together with routine hematological and biochemical tests while the appendix was collected after the surgical process. The majority of the harvested tissue was immediately refrigerated and then stored at −20°C for further biochemical assays while the remaining part of it was fixed in 10% buffered formalin for histopathological studies by the standard H and E staining. A pathologist unaware of the clinical diagnosis examined the specimens and categorized them as either acute focal, acute suppurative, acute gangrenous, and perforated appendix (Benjamin and Patel 2002).
The tissue samples were analyzed by investigators (ARS) who was unaware of the clinical/histopathological grading. Briefly, a 1 g sample of tissue was put in 10 mL of ice-cold potassium phosphate buffer (pH 7.0) and homogenized in a mechanical homogenizer (Remi Pvt., Ltd, India) in cold room. The homogenate solution was centrifuged at 10,000 × g for 15 min in a cold centrifuge. The supernatant was collected and stored at −20°C for protein content assay, GSH, LPX, and LDH. On the day of the analysis, the samples were thawed and used for various assays. All the biochemical assays were done in ultraviolet-visible spectrophotometer of Shimadzu. Tissue homogenates were assayed for total protein levels by the method of Lowry's, using bovine serum albumin as a standard  in accordance to the standard procedures performed in clinical laboratories. The levels of GSH, LPx, and LDH were estimated in tissue homogenates as follows.
Estimation of lipid peroxidation
Malondialdehyde (MDA), the sensitive and convenient marker of LPx, was assayed as thiobarbituric acid-reactive substances (TBARS) by the method of Ohkawa et al.  MDA reacts with thiobarbituric acid (TBA) at 100°C in acidic medium to form pink colored complex. The color intensity of MDA-TBA complex is measured at 535 nm. To 0.75 mL of plasma/tissue homogenate, 3 mL of MDA reagent (75 mg TBA + 15 g trichloroacetic acid, in 2.08 mL of 0.2 N HCl, made up to 100 mL with distilled water) was added, mixed and kept in boiling water bath for 20 min. Then, cooled under tap water, centrifuged at 3000 rpm for 10 min, and absorbance was measured at 535 nm against reagent blank. The MDA level was expressed in nmoles/mg protein from the standard graph plotted with different concentrations of MDA standard (1, 1, 3, 3-tetramethoxypropane procured from Sigma-Aldrich).
Estimation of glutathione
The GSH levels were estimated by the method described by Beutler et al.  GSH reduces 5, 5/dithio, bis-nitrobenzoic acid (DTNB) to yellow color 5-thionitrobenzoic acid (TNB). Absorbance measured at 412 nm is directly proportional to the concentration of GSH. Plasma/tissue homogenate was deproteinized with 2% metaphosphoric acid, and an aliquot of the supernatant was treated with DTNB and 0.3 M Disodium phosphate solution. The yellow color obtained was measured at 412 nm against the reagent blank. GSH standards ranging in concentration from 25 to 100 mg/dl were run simultaneously, and the GSH level was calculated from the standard curve and expressed as μmoles/mg protein.
Estimation of lactate dehydrogenase
The assay for LDH was performed by the kinetic spectrophotometric method described by Demetriou et al.,  using the reagent kit of Roche diagnostics. The assay is based on LDH-catalyzed reduction of pyruvate with NADH to form NAD + . The rate of oxidation of NADH to NAD + was measured as a decrease in absorbance at 340 nm and expressed in terms of units/mg protein. Internal and external quality control program of biorad was used to ensure accuracy and precision of plasma LDH values.
The values were expressed as mean with standard deviation. Significance of the difference of the values between the groups was evaluated by analysis of variance (ANOVA) and Bonferroni multiple comparison, and correlation was analyzed by Karl Pearson's Correlation Analysis using Statistical analysis software package 23 (IBM SPSS Analytics, USA).
| Results|| |
The study population consisted of 20 females and 14 men with appendicitis, whose age ranged from 12 to 48 years (mean age of 24.93 ± 8.5). The appendicitis cases were classified as per the histopathological features and the details are mentioned in [Table 1]. There were four more volunteers with normal appendix but requiring GI surgery for early colon cancer and willing for complementary appendectomy. The values of LDH, GSH, and LPx in appendicitis subjects are shown in [Table 2] and [Figure 1]. The ANOVA test showed significant differences between the different grades of appendicitis and was statistically significant LDH (P > 0.001), GSH (P > 0.001), and LPx (P > 0.002) [Figure 1] and [Table 2].
In the case of GSH, it was observed that with respect to correlation between the levels of GSH, LPx, and LDH with the histopathological grading, the results showed that a negative association of r = −0.682 for GSH; a positive association of r = 0.633 for LPx; and negative correlation of r = −0.682 for LDH [Figure 2]. Correlation of GSH, LPx, and LDH with the Alvarado grading showed a negative association of r = −0.514; a positive association of r = 0.438 for LPx; and negative correlation of r = −0.428 for LDH [Figure 3].
|Figure 2: Graphs show correlation between various biochemical parameters with histopathological grading|
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|Table 2: Levels of lactate dehydrogenase, glutathione, and malondialdehyde in tissue|
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|Figure 3: Graphs show correlation between various biochemical parameters with the clinical features (Alvarado grading)|
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Multiple regression analysis for GSH, LPx, and LDH with histopathological grading shows LDH to have a negative effect (β = −0.565, P 0≥ 0.005) followed by LPx (β = +0.283, P ≥ 0.038) and GSH (β = −0.74, P ≥ 0.689). These parameters contributed 67.1% toward correlation with the histopathological grading (R2 = 0.671) [Table 3]. With respect to the analysis of the biochemical endpoints estimated in the tissue with clinical grading, the correlation was insignificant (R2 = 0.301) and contributed only 30.1% [Table 3].
|Table 3: Results of the regression analysis for the correlation of glutathione, lipid peroxidation, and lactate dehydrogenase levels in the tissue with the histopathological and clinical grading (Alvarado grading)|
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| Discussion|| |
Oxidative stress, which refers to a condition of imbalance in oxidant-antioxidant balance due to increased generation of free radicals and impaired functioning of antioxidants, is implicated in the etiopathogenesis of many clinical conditions. , In the current study, it was observed that the levels of the cell's principal antioxidant, the GSH, was significantly decreased and that of the LPx, a marker of oxidative marker was significantly increased and correlated with the clinical and histopathological grades of severity. , These observations are in agreement to earlier reports where reports do indicate that antioxidant capacity , and total thiols  were decreased in the blood of people with acute appendicitis and also that indicators of stress such as nitric oxide, TBARS, and C-reactive protein were increased in patients with acute appendicitis. ,,
GSH is very important nucleophilic molecule and scavenges the reactive species of both oxygen and nitrogen.  It is also a cosubstrate for the antioxidant enzyme GSHPx and for the GST-mediated phase II detoxification reactions.  In addition, depletion of GSH initiates LPx which then progressively leads to severe cellular damage and cell death.  Further, it was also observed that the levels of LDH were decreased suggesting that the enhanced cell damage and increased membrane permeability possibly caused leakage of cellular enzyme into the plasma. Together, all these observations clearly indicate that the reduction in the levels of GSH enhances LPx and cell death and release of LDH. Thus, in our study, we conclude that the levels of LPx were high and that of LDH and GSH were significantly lower in the tissue as the histopathological and Alvarado scoring worsened with negative correlation for GSH and positive correlation for LPx was observed. With respect to LDH, a negative correlation for tissue was observed with both histopathological and clinical grading.
We would like to thank Dr. Malathi Bhat (Head of Biochemistry), Dr. Erel Diaz (Head of General Surgery), and Dr. Hilda Fernandes (Department of Pathology) for their support. We are also grateful to Rev. Fr. Patrick Rodrigus (Director), Rev. Fr. Denis D'Sa (Administrator), Dr. Sanjeev Rai (Former Dean), and Dr. Jayaprakash Alva, (Dean) of Father Muller Medical College for providing the necessary facilities and support. We are in debt to the patients and volunteers for providing sample for this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]