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24 May 2024: Clinical Research  

Lipid Peroxidation, Reduced Glutathione, and Glutathione Peroxidase Levels in Intervertebral Discs of Patients with Lumbar Degenerative Disc Disease

Noppawan Phumala Morales1ACEF, Witcharat Loahachanwanich2B, Ekkapoj Korwutthikulrangsri2AE, Monchai Ruangchainikom2AE, Werasak Sutipornpalangkul2ABDEF*

DOI: 10.12659/MSM.944335

Med Sci Monit 2024; 30:e944335

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Abstract

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BACKGROUND: Either a reduction in antioxidant levels or an accumulation of reactive oxygen species can heighten susceptibility to oxidative damage in disc cells. To date, no research has investigated the levels of lipid peroxidation products (thiobarbituric acid reactive substances [TBARs]), reduced glutathione (GSH), and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products in excised disc tissues from patients with degenerative disc disease.

MATERIAL AND METHODS: Forty-two patients were enrolled. Patients were divided into lumbar disc degeneration (LDD) and nonlumbar disc degeneration (nonLDD) groups according to Pfirrmann classification. Intervertebral discs were obtained from all patients during the operation and were homogenized for analysis. TBARs levels were measured using fluorometry. GSH levels and GPx activity were quantified spectrophotometrically using a kinetic method.

RESULTS: TBARs levels in excised discs from LDD patients (5.18±4.14) were significantly higher than those from nonLDD patients (2.56±1.23, P=0.008). The levels of TBARs tended to increase with the severity of degeneration according to the Pfirrmann classification. However, these 2 groups showed no significant differences in reduced glutathione levels or glutathione peroxidase activity (P>0.05). Patients with LDD exhibited a worse health-related quality of life, reflected in lower utility and EQ-VAS scores and higher Oswestry disability index scores.

CONCLUSIONS: There was a notable increase in lipid peroxidation products in the excised intervertebral discs of patients with LDD. This finding suggests that oxidative stress may contribute to the development of disc degeneration.

Keywords: Glutathione, Glutathione Peroxidase, intervertebral disc degeneration, Lipid Peroxidation, Oxidative Stress

Introduction

Low back pain has emerged as a profoundly debilitating and increasingly common condition, imposing substantial social and economic burdens globally due to lost income and reduced productivity [1]. While the etiology of low back pain is diverse, lumbar intervertebral disc degeneration is frequently identified as a primary contributor [2,3].

Numerous studies have highlighted oxidative stress as a key factor in the development of chronic degenerative diseases, such as knee osteoarthritis [4,5]. In the context of lumbar discs, oxidative stress is implicated in matrix deterioration and cell apoptosis, potentially accelerating disc degeneration [6–8]. Limited evidence suggests that either a reduction in antioxidant levels in degenerative discs or an accumulation of reactive oxygen species heightens susceptibility to oxidative damage in disc cells [9,10]. This degenerative process has been linked not only to localized oxidative stress but also to systemic oxidative stress, a phenomenon that has also been associated with intervertebral disc degeneration [11].

Previous research has documented the extent of oxidative damage to lipids in degenerative disc diseases. Thiobarbituric acid reactive substances (TBARs), a by-product of lipid peroxidation, have been observed to accumulate in rat plasma and lumbar intervertebral discs [9]. Elevated TBARs levels were also reported in human plasma from patients with disc degeneration compared to healthy individuals [12,13]. Furthermore, lipid peroxidation levels were found to be substantially higher in human herniated disc tissues than in control tissues [14], and a similar increase was noted in the plasma of individuals with disc herniation [15].

The current understanding of antioxidant status in degenerative disc disease remains limited and subject to debate. Hou et al observed a decrease in superoxide dismutase levels in the plasma and lumbar intervertebral discs of rats due to age-related changes [9], a trend also noted in human plasma in cases of disc herniation [11]. Conversely, other studies have reported conflicting results concerning age-related variations in superoxide dismutase activity, with increases observed in healthy Chinese adults [16,17]. Reduced glutathione (GSH) activity has also been found to be diminished in patients with disc herniation [11]. In addition, plasma levels of glutathione reductase have been reported to be markedly lower in patients with degenerative disc disease than in healthy individuals [12].

To date, no research has specifically investigated the levels of lipid peroxidation, GSH, and glutathione peroxidase (GPx) in excised human lumbar disc tissues affected by degenerative disease. Therefore, this study aimed to evaluate lipid peroxidation products of oxidative stress in the excised lumbar vertebral disc tissues of patients with degenerative lumbar disc degeneration.

Material and Methods

ETHICS APPROVAL:

This cross-sectional study was conducted at the Department of Orthopedic Surgery of Siriraj Hospital, Mahidol University, Bangkok, Thailand. Data were sourced from the hospital’s electronic medical records and the Siriraj Spine Registry database. Approval for the study was granted by the Ethics Committee of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. Si-543/2020). The guidelines and regulations of the Ethics Review Board were followed for all material and methods. Written informed consent was obtained from all participants prior to data and sample collection.

PARTICIPANTS:

We recruited 42 adult patients between the ages of 18 and 80 who underwent single-level lumbar spinal surgery, such as discectomy or spinal fusion, at Department of Orthopaedics, Faculty of Medicine Siriraj Hospital. Patients with a history of spine surgery, revision spinal surgery, spinal infections, or spinal tumors were excluded.

The sample size calculation was based on the data reported by Hou et al regarding TBARs levels and age-related changes in rat lumbar intervertebral discs [9]. The TBARs levels in young and geriatric rats were reported as 5.46 and 8.99, respectively. The sample size calculation was performed using the Statulator online tool (https://statulator.com/). Assuming 80% power and a 5% significance level (two-sided), we determined that a sample size of at least 10 patients per group was necessary for the study.

GRADING SYSTEM FOR LUMBAR INTERVERTEBRAL DISC DEGENERATION:

Our study categorized patients into 2 groups based on their Pfirrmann classifications [18]. Patients with discs classified as Pfirrmann grades I or II were placed in the nonlumbar disc degeneration (nonLDD) group. Those with discs graded as Pfirrmann grades III, IV, or V were categorized as having lumbar disc degeneration (the LDD group). Grade I is characterized by a homogeneous disc with high bright white signal intensity and a normal disc height. Grade II has an inhomogeneous disc structure with high white signal intensity and the distinction between the nucleus and annulus is clear (Figure 1A). Grade III is the same as in Grade II, with an inhomogeneous disc structure with intermediate gray signal intensity, but the distinction between the nucleus and annulus is unclear, and the disc height is normal or moderately decreased (Figure 1B). Grade IV indicates an inhomogeneous disc with low dark gray signal intensity and disc height is slightly or moderately low (Figure 1C). Grade V is characterized by an inhomogeneous disc structure with low back signal intensity; there is no difference between the nucleus and annulus, and the disc space is collapsed (Figure 1D). Three spine surgeons (EK, MR, and WS) assessed the degree of disc degeneration using T2-weighted neutral sagittal images to assign one of 5 grades following the criteria established by Pfirrmann et al.

LUMBAR INTERVERTEBRAL DISC COLLECTION:

A segment of the lumbar intervertebral disc, weighing 0.5–1 g, was collected during each discectomy or interbody spinal fusion procedure (posterior lumbar interbody fusion, transforaminal lumbar interbody fusion, or extreme lateral interbody fusion). The samples were immediately irrigated with normal saline to eliminate blood cell contamination and stored at −80°C until analysis.

LUMBAR INTERVERTEBRAL DISC PREPARATION:

Each disc sample was finely chopped using sterile scissors, and 1 mL of phosphate-buffered saline was added. Homogenization was performed using a lysing homogenizer with stainless steel beads (Ohaus HT Lysing Bead Mill Homogenizer, Parsippany, NJ, USA) at 1200 rpm for 5 cycles of 1.3 minutes each. The homogenate was centrifuged using a microcentrifuge (Spectrafuge 24D; Labnet International, Inc., Woodbridge, NJ, USA) at 10 000 rpm for 10 minutes. The supernatant was then extracted for GSH and GPx assays, while the entire homogenate was utilized for lipid peroxidation assays.

DETERMINATION OF LIPID PEROXIDATION PRODUCTS (TBARS):

Lipid peroxidation was assessed by measuring TBARs levels in the samples using a fluorometric method described by Asakawa and Matsushita et al [19]. Briefly, the 500 μl of homogenate was diluted with PBS buffer to 2 ml then added 100 μl of 100 mM butylated hydroxytoluene (BHT), 1 ml of 10% trichloroacetic acid (TCA), 500 μl of 5 mM ethylene diamine tetraacetic acid (EDTA), and 250 μl of 8% sodium dodecyl sulfate (SDS) in sequence. Finally, a 750 μl of 0.6% thiobarbituric acid (TBA) was added before heating at 90°C for 1 hour. After cooling, the mixture was centrifuged at 1690 g for 10 min (Flexpin Bench-top centrifuges, TOMY-LC200, CA). The fluorescence intensity of clear pink supernatant was measured using a multimode microplate reader (Varioskan Flash Microplate Reader, Thermo Scientific, USA) with excitation and emission wavelengths at 515 and 553 nm, respectively. We used 1, 1, 3, 3-tetraethoxypropane as the standard.

DETERMINATION OF GSH LEVELS:

Total GSH concentration was measured using the DTNB-glutathione reductase recycling method according to the method of Anderson et al [20], using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) as the disulfide chromogen that is easily reduced by sulfhydryl compounds to an intensely yellow compound. Briefly, the 50 μl of supernatant was incubated with 200 μl reaction mixture (0.4 mM nicotinamide adenine dinucleotide phosphate (NADPH) and 1.2 U/ml GSH reductase in 0.01 mM sodium phosphate buffer containing 0.05 mM EDTA) at 30°C for 2 min before addition of 20 μl DTNB. The absorbance was measured at 412 nm at 30-second intervals for 3 minutes with a multimode microplate reader (Varioskan Flash Microplate Reader, Thermo Scientific, USA). The GSH concentration was calculated by comparing the slope of the 5 concentrations of standard GSH and was expressed as nmol/mg protein.

DETERMINATION OF GPX ACTIVITY:

The activity of GPx was quantified by the spectrometric methods, as described by Takahashi et al [21]. Briefly, the 20 μl of supernatant was incubated with 185 μl reaction mixture (25 mM GSH and 0.2 mM NADPH in 0.25 M Tris-HCl containing 1.25 mM EDTA) and 25 μl of 10 U/ml GSH reductase 37°C for 2 min before addition of 20 μl of 1.75 mM t-butyl hydroperoxide (t-BuOOH). The oxidation of NADPH was monitored at 340 nm at 15 seconds interval for 10 minutes with a multimode microplate reader (Varioskan Flash Microplate Reader, Thermo Scientific, USA). The slope was obtained and used a normalized extinction coefficient (ɛ) of 2.57 mM−1 to calculate the concentration of NADPH. Intervertebral disc GPx activity was expressed as μmol NADPH/min per mg protein.

HEALTH-RELATED QUALITY OF LIFE ASSESSMENTS:

The health-related quality of life and functional outcomes of patients were assessed using the EQ-5D-5L, the EQ-VAS, and the Oswestry disability index. EQ-5D-5L scores were converted to utility scores using previously published coefficient factors specific to the Thai population [22].

STATISTICAL ANALYSIS:

The demographic and clinical characteristics of the patients were analyzed and reported descriptively. Categorical data were compared using the chi-square test and Fisher’s exact test, as appropriate. The distribution of continuous numeric data was assessed using the Shapiro-Wilk test. Normally distributed continuous data were compared using independent t tests. The results are presented as the mean±standard deviation for continuous variables and frequency (percentage) for categorical variables. Since the data were not normally distributed, nonparametric tests such as the Mann-Whitney U test or Wilcoxon signed-rank test were used for analysis. A two-tailed probability (P) value of less than 0.05 was considered statistically significant.

Results

DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF PARTICIPANTS:

The demographic and clinical characteristics of the patients with and without LDD are presented in Table 1. Patients in the LDD group were significantly older (P<0.001). There were no significant differences in sex (P=0.127) or body mass index (P=0.089) between the 2 groups. The most common diagnoses in both groups were herniated nucleus pulposus and spinal stenosis. All patients in the nonLDD group were classified under Pfirrmann grade II and had no underlying diseases.

LIPID PEROXIDATION PRODUCTS AND HEALTH-RELATED QUALITY OF LIFE:

Table 2 showed the TBAR, GSH, and GPx levels in the lumbar intervertebral discs of the patients with and without LDD. A significant difference was found in TBAR levels in the LDD group (5.18±4.14 nmol/g tissue) compared to the nonLDD group (2.56±1.23 nmol/g tissue, P=0.008). TBAR levels tended to rise as the severity of degenerative discs increased (higher in Pfirrmann grades IV and V than in grade III), although this trend did not reach statistical significance (Figure 2). GPx and GSH levels showed no significant differences between the patient groups (P=0.299 and 0.708, respectively).

As detailed in Table 3, the LDD group exhibited a worse health-related quality of life than the nonLDD group. This difference was evidenced by the LDD group’s lower utility scores (P=0.001), higher Oswestry disability index scores (P=0.005), and lower EQ-VAS scores (P=0.007). No correlation was observed between TBAR levels and quality of life.

Discussion

STRENGTHS AND LIMITATIONS:

Our study has several strengths. First, it drew upon data from an earlier study to calculate the sample size needed to evaluate differences in TBARs levels. Second, it included a larger patient cohort than in prior reports. Third, while previous studies primarily focused on cell cultures and animal models, our study utilized excised human intervertebral discs. This approach allowed us to demonstrate oxidative stress in local tissues and may shed light on the mechanisms of oxidative stress in disease lesions. However, a limitation of our study is the potentially inadequate concentration of antioxidants and antioxidant enzymes in the homogenate samples. For instance, the α-tocopherol and nitric oxide levels were below detectable limits.

Conclusions

Our study demonstrated that patients with degenerative lumbar discs exhibited higher oxidative stress levels. Based on this finding, we propose that oxidative stress may play a role in the development of LDD, and antioxidant therapy may have the potential to slow the progression of LDD.

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