Comparative Analysis of Carbonic Anhydrase-II Expression Across Undescended Testes Sprague Dawley Model with Coenzyme Q10 Treatment

Abstract

Background:

The study investigates the expression of Carbonic Anhydrase-II (CA-II) antibodies in both blood and tissue samples to understand their systemic and local effects.

Objective:

The research aims to identify effective dosages and assess the differences in antibody levels across various treatment groups of undescended testes Sprague Dawley model. To measure and compare the levels of CA-II antibodies in blood and tissue samples, determine the most effective dosage for reducing tissue CA-II levels, and analyze the systemic versus local impacts of these antibodies.

Methods:

The study employed immunohistochemistry to assess CA-II antibody expression in undescended testes model of Sprague Dawley tissue and blood, with brown-colored cells indicating positive expression. Levene’s test confirmed homogeneity of variance (p=0.660), allowing for ANOVA to identify significant differences in CA-II levels among groups (p=0.000). Tukey’s post hoc test was used to pinpoint specific group differences. Results: The analysis revealed significant differences in CA-II expression between groups. The dosage of 10 mg/KgBB was found to be most effective in reducing tissue CA-II levels. Blood CA-II concentrations were consistently higher than tissue levels across all groups, indicating a strong systemic presence.

Conclusion:

The study highlights the significant distinctions in CA-II antibody levels between blood and tissue samples. It underscores the importance of dosage in managing CA-II levels and the need to consider both systemic and local impacts in clinical settings. These findings provide a basis for future research into targeted therapies for conditions mediated by CA-II.

1. BACKGROUND

Carbonic Anhydrase II (CA-II) is a zinc metalloenzyme that facilitates the reversible conversion of carbon dioxide (CO₂) into bicarbonate (HCO3−) and protons (H+), playing a crucial role in pH regulation, CO2 transport, and maintaining acid-base equilibrium (1, 2). Though predominantly located in red blood cells, CA-II is also found in the kidneys, brain, and various other tissues, underlining its broad physiological relevance (1, 2). Its rapid catalytic turnover, reaching approximately 106 reactions per second, highlights its essential function in numerous metabolic activities (3).

In addition to its physiological roles, CA-II has been associated with several pathological conditions. Disorders such as osteopetrosis and renal tubular acidosis have been linked to genetic mutations in the CA2 gene, underscoring its importance in skeletal and renal health (4). Variations in CA-II expression have also been noted in certain cancers, where the enzyme may influence tumor aggressiveness and progression (5). In reproductive health, CA-II is important for maintaining ion and pH balance within the testicular microenvironment. Disruptions in CA-II activity have been implicated in cryptorchidism (undescended testes), a condition associated with infertility and a heightened risk of testicular cancer (6, 7).

A comparative analysis of CA-II expression in tissue and blood samples across different treatment groups is essential for understanding the enzyme’s systemic versus local effects. Therefore measuring CA-II both in tissue and blood accurately is a crucial step for making it reliable biomarker in clinical diagnostics and research. Even though presents challenges to standardizing measurement protocols can be influenced by sample properties and assay conditions (8, 9). This modalities are suggested to offer enhanced sensitivity and specificity, as they require further proof for broad clinical application (10, 11).

2. OBJECTIVE

This study aims to measure the expression levels of Ca-II antibodies in blood and tissue samples across different treatment groups to distinguish between systemic and local effects. By examining the differential expression of CA-II, the study aims to elucidate its role in both physiological and pathological contexts, ultimately contributing to improved therapeutic strategies and patient outcomes.

3. MATERIAL AND METHODS

Study design

A controlled experimental approach was utilized to explore the relationship between Carbonic Anhydrase II (CA-II) concentration in tissue and blood using an ELISA framework. The primary objective was to develop a reliable quantitative method for accurately measuring CA-II, supporting its application as a biomarker in both clinical and research environments.

The study utilized six treatment groups using Sprague Dawley, consisting of:

• Non-UDT group (Negative Control);

• Unilateral UDT group without orchidopexy (Positive Control);

• Unilateral UDT group with orchidopexy (P1);

• Unilateral UDT group with orchidopexy followed by Coenzyme-Q10 at a dose of 5 mg/kg for 7 days (P2);

• Unilateral UDT group with orchidopexy followed by Coenzyme-Q10 at a dose of 10 mg/kg for 7 days (P3);

• Unilateral UDT group with orchidopexy followed by Coenzyme-Q10 at a dose of 20 mg/kg for 7 days (P4).

Sample preparation

The preparation of samples involved the use of purified human CA-II protein as a standard. A series of dilutions were performed to generate CA-II concentrations ranging from 0 ng/mL (blank control) to 100 ng/mL, covering the physiological levels expected in clinical specimens. Each concentration was divided into 100 μL aliquots and stored at -20°C to preserve stability and prevent degradation from repeated freeze-thaw cycles. Prior to the assay, aliquots were thawed on ice and gently vortexed to ensure uniformity.

ELISA protocol

The ELISA procedure began with high-binding 96-well microtiter plates (Nunc MaxiSorp) being coated with 100 μL of anti-human CA-II capture antibodies, diluted to 1 μg/mL in a carbonate-bicarbonate buffer (pH 9.6). Plates were left to incubate overnight at 4°C to ensure effective antibody binding. The following day, plates underwent three washes with 300 μL of PBS containing 0.05% Tween-20 (PBST) to eliminate unbound antibodies. Blocking was achieved using 200 μL of 5% non-fat dry milk in PBS for 1 hour at room temperature to minimize non-specific binding, followed by three additional PBST washes.

Subsequently, 100 μL of CA-II standards or test samples were introduced to each well in duplicate and incubated at 37°C for 2 hours with gentle shaking to facilitate antigen-antibody interaction. After washing, 100 μL of biotinylated anti-CA-II detection antibody, prepared at 0.5 μg/mL in PBS with 0.1% BSA, was added and incubated for 1 hour at room temperature. Plates were washed again three times, followed by the addition of 100 μL of Streptavidin-HRP, prepared according to the manufacturer’s guidelines, and incubated for 30 minutes at room temperature. Five washes were performed to ensure the complete removal of unbound conjugate.

For color development, 100 μL of TMB substrate was added to each well and incubated in the dark at room temperature for 15 minutes. The reaction was halted by adding 50 μL of 2M sulfuric acid, causing the color to shift from blue to yellow. OD measurements were taken at 450 nm, with 620 nm as the reference wavelength, using a microplate reader within 30 minutes to ensure accuracy.

Immunohistochemistry of CA-II

Immunohistochemistry was employed to visualize CA-II expression in tissue samples. Tissue sections were first deparaffinized and rehydrated through a graded series of alcohols. Antigen retrieval was performed using a citrate buffer (pH 6.0) heated to 95°C for 20 minutes. Following this, sections were allowed to cool to room temperature. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide in methanol for 10 minutes. Non-specific binding sites were then blocked using a 5% bovine serum albumin (BSA) solution for 30 minutes.

Primary anti-CA-II antibodies were applied to the sections and incubated overnight at 4°C in a humidified chamber. The following day, sections were washed with PBS and incubated with a biotinylated secondary antibody for 30 minutes at room temperature. After washing, the sections were treated with an avidin-biotin complex (ABC) reagent for 30 minutes. The antigen-antibody complex was visualized using a diaminobenzidine (DAB) substrate, resulting in a brown color indicating positive CA-II expression. Finally, sections were counterstained with hematoxylin, dehydrated, and mounted for microscopic examination.

Data analysis

Raw data readings were exported from the microplate reader into a CSV file for statistical evaluation. The average data in ng/mL for each standard and test sample (measured in duplicate) was calculated to maintain consistency. Linear regression analysis was conducted to construct a standard curve and derive the calibration equation along with the R2 value, establishing the relationship between CA-II concentration. The Shapiro-Wilk test was applied to confirm the normality of data distributions.

To compare data values across multiple sample groups, One-Way ANOVA was performed. When significant differences were identified, Tukey’s HSD post-hoc test was used to pinpoint specific group differences. Pearson correlation analysis assessed the strength and direction of the relationship between CA-II concentration each group. Data analysis was conducted using Python libraries (pandas, SciPy, statsmodels) and SPSS (version 11.0) to ensure robust statistical assessment.

To promote reproducibility, standardized reagent preparation and consistent incubation conditions were employed throughout the experiments. Each assay was performed at least three times to validate the reliability of the results. Detailed documentation, including batch numbers of reagents and equipment calibration logs, was maintained to facilitate replication by other researchers.

4. RESULTS

In this study, the expression of Ca-II antibodies was measured in different samples to evaluate systemic and local effects. Blood samples were analyzed to assess systemic effects, while tissue samples were examined to determine local effects.

Tissue Ca-II expression

The highest tissue expression was observed in group KP (12.66 ± 2.16 ng/mL), while group P4 showed the lowest expression (5 ± 1.41 ng/mL) as showed in Figure 5.1 and 5.2. Homogeneity of variance for tissue Ca-II data among groups N, KP, P1, P2, P3, and P4 was assessed using Levene’s test, yielding a significance value of 0.660, which is greater than 0.05. This indicates that the variance in tissue Ca-II levels among these groups is homogeneous. Consequently, post hoc tests such as Tukey HSD, LSD, and Bonferroni can be applied following ANOVA, as the data meet the assumption of homogeneity.

ANOVA results show a p-value of 0.000 (p < 0.05), leading to the rejection of the null hypothesis (Ho) and acceptance of the alternative hypothesis (H1). This confirms significant differences in tissue Ca-II expression among groups N, KP, P1, P2, P3, and P4. Given these significant differences, a post hoc analysis using Tukey’s test was conducted.

Table 5.3 Post hoc CA-II expression in tissue

The post hoc Tukey test revealed that tissue Ca-II expression in group N significantly differed from that in groups KP and P1 (p < 0.05), but not from groups P2, P3, and P4. Tissue Ca-II expression in group KP significantly differed from that in groups P2, P3, and P4 (p < 0.05), but not from group P1. Tissue Ca-II levels in group P1 significantly differed from those in groups P3 and P4 (p < 0.05), but not from group P2. No significant .ifferences were observed between groups P2, P3, and P4. These findings suggest that Q10 affects the reduction of tissue Ca-II, with effective doses being 10 mg/KgBB and 20 mg/KgBB, starting with an initial dose of 10 mg/KgBB.

Blood Ca-II Levels

The highest blood expression was found in group P1 (23.85 ± 1.96 ng/mL), and the lowest was in group N (20.73 ± 3.27 ng/mL). The post hoc Tukey test results indicate that blood Ca-II levels in group N significantly differed from those in groups KP and P1 (p < 0.05), but not from groups P2, P3, and P4. Blood Ca-II levels in group KP significantly differed from those in groups P2, P3, and P4 (p < 0.05), but not from group P1. Blood Ca-II levels in group P1 significantly differed from those in groups P3 and P4 (p < 0.05), but not from group P2. No significant differences were observed between groups P2, P3, and P4.

Figure 1. The immunohistochemistry results for Ca-II antibodies, where brown-stained cells indicate positive expression of Ca-II antibodies, and blue-stained cells represent non-expressing cells.

Figure 2. The levels of Ca-II expression in tissue across different groups.

Correlation between tissue and blood Ca-II expression

The comparison between Figures 5.2 and 5.3 reveals that blood Ca-II levels are consistently higher than tissue levels across all treatment groups. This suggests a stronger systemic presence of Ca-II relative to its local expression in tissues. The significant differences in expression levels between blood and tissue highlight the distinct roles and impacts of Ca-II in systemic versus local environments. This association underscores the importance of considering both blood and tissue measurements when evaluating the effects of treatments on Ca-II expression.

Figure 3. The levels of Ca-II in blood across the different groups.

Figure 3. The levels of Ca-II in blood across the different groups.

5. DISCUSSION

The findings of this study reveal significant differences in Carbonic Anhydrase II (CA-II) expression between tissue and blood samples across various treatment groups. The elevated levels of CA-II in the blood compared to tissue suggest a prominent systemic presence, which aligns with previous research highlighting CA-II’s widespread physiological roles (1, 2). This systemic expression is crucial for maintaining pH balance and facilitating CO₂ transport, as CA-II is predominantly found in red blood cells but also expressed in other tissues like the kidneys and brain (1, 2).

The significant differences observed in tissue CA-II expression among treatment groups may be attributed to the varying impacts of orchidopexy and Coenzyme-Q10 administration. The highest tissue expression was noted in the KP group, indicating that unilateral UDT without treatment may lead to increased local CA-II levels. This finding underscores the enzyme’s role in local tissue environments, possibly linked to pathological conditions such as cryptorchidism (6, 7).

Coenzyme-Q10’s effect in reducing tissue CA-II levels, particularly at doses of 10 mg/kg and 20 mg/kg, suggests its potential therapeutic benefit in managing conditions associated with altered CA-II expression. This aligns with studies exploring CA-II activators and inhibitors in various health conditions, including osteopetrosis and certain cancers (4, 5).

The use of statistical methods like ANOVA and Tukey’s post hoc test was essential in identifying these significant differences, ensuring robust data analysis and interpretation. The homogeneity of variance confirmed by Levene’s test further validates the reliability of these findings. This further enforces previous study under the same roadmap (12).

Overall, this study contributes to the understanding of CA-II’s dual role in systemic and local environments, emphasizing the need for targeted therapeutic strategies to modulate its expression effectively. Future research should continue to explore the molecular mechanisms underlying CA-II’s involvement in both physiological and pathological processes, potentially leading to improved clinical outcomes and personalized treatments (8, 9).

6. CONCLUSION

This study provides valuable insights into the differential expression of Carbonic Anhydrase II (CA-II) in blood and tissue samples across various treatment groups. The consistently higher CA-II levels in blood compared to tissue highlight its significant systemic presence and potential role in broader physiological processes. Coenzyme-Q10 demonstrated efficacy in reducing tissue CA-II levels in dose effective manner. These conclusions contribute to a deeper understanding of CA-II’s function and pave the way for future research into targeted therapeutic interventions.

Bullet Points

• CA-II levels were consistently higher in blood than in tissue, highlighting its significant systemic presence and role in physiological processes.

• Effective in reducing tissue CA-II levels at doses of 10 mg/kg and 20 mg/kg, suggesting its potential as a therapeutic agent.

• Significant differences in CA-II expression among treatment groups were confirmed using ANOVA and Tukey’s post hoc test, ensuring the study’s reliability.

Authors’ contributions:

The all authors were involved in all steps of preparation this article, including final proofreading.

Conflict of interest:

The authors have no conflict of interest to declare.

Financial support and sponsorship:

This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors.