The study was carried out in compliance with the Declaration of Helsinki and approved by the Institution Ethics Committee of SGT University, Faculty of Medicine and Health Sciences, with vide letter number IEC/FMHS/MD/MS/2023-29 dated 03/06/2023. Written and informed consent was obtained after explaining the purpose and details of the study to all of the subjects. Protocol numbers were assigned for reference, and participant personal information was kept private and used only for research. Data security measures included password protection of the computer and data storage in a safe physical location.

This observational cross-sectional study was carried out in the Department of Biochemistry of the Faculty of Medicine and Health Sciences at SGT University, Gurugram, India, from 01 July 2023 to 30 June 2024.

Acne severity was assessed by a single experienced dermatologist using a simple grading system by Tutakne and Chari,²¹ to ensure consistency and avoid interrater variability:

By keeping the confidence level at 95% (1.96), prevalence (85%),²² and maximum allowance of error (5%) in the Cochran’s formula (n = Z²pq/e²), a sample size of 196 subjects was established. We rounded up to 200 to account for potential exclusions. Therefore, 200 subjects (50 in each grade of AV: Grades 1, 2, 3, and 4) and 50 healthy controls were selected. For sub-group comparison, a post-hoc power calculation was performed in acne grades. By keeping alpha = 0.05, power (1-β) comes as: for SOD, large effect size (Cohen’s d = 1.14) and power = 1.0. Malondialdehyde and GSH (glutathione) comparisons demonstrated high power, whereas CAT comparisons had smaller effect sizes and slightly lower power (0.85).

The cross-sectional study included patients with AV, who did not receive any topical or systemic treatment in the previous four weeks, were aged between 18 and 45 years, were from both genders, and were willing to participate. However, the study excluded patients on immune-suppressive therapy such as corticosteroids, regular analgaesic intake, and hormonal therapy; patients with any systemic disease (diabetes mellitus, liver disease, thyroid disorders, cardiovascular events, known cases of malignancy); patients with any dermatological disorder other than AV; patients taking any vitamin supplementation; pregnant and lactating women; and smoking and/or alcohol abusers. The information on social demographics and disease duration was collected by directly interviewing study participants in the local language using a semi-structured questionnaire.

Using aseptic technique, 5 mL of venous blood was taken in dry sterile red top (serum) vacutainers from each participant after gaining written consent. Serum was separated by centrifugation at 3500 revolutions per minute (rpm) for 10 min. Serum SOD, CAT, GSH and MDA were measured using the ERBA Lisa Scan EM analyser (ERBA Diagnostics Mannheim GmbH, Mannheim, Germany) via an enzyme-linked immunosorbent assay kit. Calibration and quality control of this analyser were performed before the samples were run. The manufacturer’s instructions for the machine and the reagents were strictly followed. Standard curves were generated for each biomarker (SOD, CAT, GSH, MDA) using the calibrators provided in the enzyme-linked immunosorbent assay kits, and sample concentrations were calculated from these curves. To reduce the analytical variation, all samples were analysed using the same lots of reagent kits, and intra-assay coefficient of variation values remained within acceptable limits. (Table 1).

Data and various parameters were analysed on IBM^® SPSS^® Statistics (SPSS^®) version 24 (IBM Corporation, Armonk, New York, United States). The Shapiro-Wilk test was applied for assessing the normal distribution of the dataset and was found to be normally distributed. Mean and standard deviation of all parameters were calculated. A Student’s t-test (unpaired) was used to compare two groups while a one-way analysis of variance test was applied to compare the various parameters in three or more groups. A post-hoc test was conducted to determine pair-wise differences between groups. Pearson’s correlation coefficient (r-value) was applied to find the correlation. The p < 0.05 was considered as statistically significant, and p < 0.001, highly significant.

The present study included 50 healthy controls: 29 women and 21 men with a mean age of 25.80 ± 5.45 years. Two hundred patients with AV—121 females and 79 males—were also enrolled (Figure 1). The mean age and gender distribution of each grade of AV is expressed in Table 2, with p = 0.0007. The distribution of patients according to duration of acne is shown in Table 3. Over 31.4% of the women had struggled with AV for 2–5 years, while a higher number of men (50.6%) had acne for a similar time period, followed by 34.2% who had acne for more than five years (p < 0.0001).

Table 4 shows a comparison of SOD, CAT, GSH and MDA levels between controls and severity grades of acne. The serum levels of SOD were lowest in severe cases (Grade 4) and highest in mild cases (Grade 1), when compared with controls with a mean ± standard deviation of 5.68 U/mL ± 1.95 U/mL, 11.71 U/mL ± 2.12 U/mL, and 89.39 U/mL ± 9.91 U/mL (p < 0.0001). Similarly, the serum levels of CAT and GSH were markedly decreased in Grade 4 acne patients versus individuals in any other grades. Meanwhile, a comparison of MDA levels across the four grades revealed a substantial difference. Malondialdehyde levels were significantly lower in Grade 1, and higher in Grades 3 and 4 (p < 0.0001). Post-hoc pair-wise comparisons between acne severity grades revealed significant differences for most oxidative stress markers. Superoxide dismutase levels showed highly significant reductions across all grade comparisons (p ≤ 0.0001). Catalase levels were comparable between Grades 1 and 2 (p = 0.0703), but significantly lower from Grade 2 onwards (p ≤ 0.0001). Glutathione levels demonstrated an early decline, with a significant difference observed between Grades 1 and 2 (p = 0.0161), and highly significant differences in subsequent grade comparisons (p < 0.0001). Malondialdehyde levels were significantly elevated across all inter-grade comparisons (p < 0.0001), confirming progressive oxidative damage with increasing acne severity (Table 5).

To adjust for potential confounding variables and to explore the combined effect of oxidative stress markers, a multiple linear regression analysis was performed. Acne severity was used as the dependent variable, with serum levels of SOD, CAT, GSH, MDA, and patient age as independent predictor variables. As shown in Table 6, MDA was a significant positive predictor (B = 0.232, p < 0.0001), while SOD (B = −0.125, p < 0.0001), CAT (B = −1.920, p = 0.0004), and GSH (B = −0.839, p < 0.0001) were significant negative predictors. Age was not a significant predictor (p = 0.311). Overall, these findings indicate that oxidative stress markers independently contribute to acne severity, with higher oxidative damage (MDA) and lower antioxidants (SOD, CAT, GSH) predicting more severe disease.

Table 7, Table 8, Table 9, Table 10, Figure 2, Figure 3, Figure 4 and Figure 5 displays the correlation between acne severity and levels of oxidative and antioxidative biomarkers. A significant negative correlation was observed between MDA levels and CAT activity in the serum of Grade 4 acne patients (r = –0.403, p = 0.0037). However, no significant correlation was found in the other acne subgroups.

The limitations of the study were as follows:

Future research should involve lesion-level assays–such as quantification of squalene peroxides in sebum, punch biopsy, or scraping of skin surface by a dermatologist for correlating serum and local cutaneous oxidative stress directly.

According to the present results, statistically high levels of MDA in patients with AV is evidence of the existence of oxidative stress which can cause cell injury and inflammation. Furthermore, low levels of SOD, CAT and GSH enzyme in acne clearly indicate that the antioxidant defence system is damaged, irrespective of the degree of severity, and thereby antioxidant drugs can be indicated for the treatment of acne. However, it is still unclear whether oxidative stress is a cause or an outcome of inflammation. Combining antioxidants drugs with other treatment modalities of acne might be beneficial in treating patients, especially those with inflammatory acne lesions, or their effects may be mitigated.