Serum adenosine deaminase, catalase, and carbonic anhydrase activities in patients with renal cell carcinoma

Abstract

Objectives: To determine whether serum levels of adenosine deaminase (AD), catalase (CAT), and carbonic anhydrase (CA) enzymes may be useful biomarkers in the diagnosis of renal tumors and may lead to early diagnosis of renal tumors.

Material and methods: The study included 33 patients with renal cell carcinoma (RCC) and 31 healthy controls. The activity of serum AD, CA, and CAT was determined and analyzed using the Giusti spectrophotometric method, H₂O₂ substrate, and C0₂ hydration, respectively.

Results: Serum AD and CA activity were significantly higher in patients with RCC than in controls. However, serum CAT activity was significantly lower in patients with RCC than in controls.

Conclusion: These markers might be potentially important as an additional biochemical tool for diagnosing RCC. We believe multidisciplinary studies are needed to plan patients’ preoperative and postoperative treatment and to create follow-up protocols.

Keywords:

• Adenosine deaminase
• Antioxidant enzymes
• Carbonic anhydrase
• Catalase
• Renal cell carcinoma

Introduction

In the United States, approximately 30 800 new cases of renal cancer and 12 000 deaths from renal cancer are reported annually, making it the sixth-largest cause of cancer deaths. Renal cell carcinoma (RCC) accounts for 90–95% of all kidney malignancies.¹ The incidence of RCC has been increasing worldwide by approximately 2–4% per year over the last 20 years. Advances in diagnostic imaging and early detection do not fully explain this trend.² Established risk factors for renal cancer include smoking, use of phenacetin-containing drugs, hypertension, obesity, and end-stage renal disease.³ Of the patients who present with localized disease and are considered for surgery with curative intent, approximately one-third will go on to develop metastatic disease. Metastatic kidney cancer is resistant to all ‘standard’ forms of radiation therapy, chemotherapy, and hormonal therapies used to treat other types of carcinomas.

Some defense mechanisms in the body prevent the development of free radicals and the damage they cause. One of the most important of these antioxidant enzymes is superoxide dismutase, which catalyzes the dismutation of superoxide anion into hydrogen peroxide (H₂O₂), which is removed by catalase (CAT), a well-known plasma antioxidant enzyme,⁴ and glutathione peroxidase.

Carbonic anhydrase (CA) is a member of the alpha family of CAs of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to carbonic acid. CAs have recently become a target of intensive research into carcinogenesis and tumor invasion.⁵

In the purine metabolic pathway, adenosine deaminase (AD) is an important aminohydrolase that catalyzes the conversion reaction of adenosine to inosine and of deoxyadenosine to deoxyinosine.⁶ The relationship between AD and various cancers has been investigated by several clinical studies. Although several studies have found increased enzyme activity in some cancerous tissue,^7–9 it has typically been found to be decreased in various neoplastic tissues.^10,11

A single study evaluating AD, CAT, and CA levels found all three of these parameters to be higher in patients with bladder cancer.¹² To the best of our knowledge, no previous study has reported serum AD, CA, and CAT activities in patients with RCC. Therefore, we aimed to simultaneously investigate serum AD, CA, and CAT activities in patients with RCC.

Materials and methods

The study included 33 patients (24 males, 9 females)with RCC. Mean age was 54.1 ± 4.3. None of the patients had ever smoked, been addicted to alcohol, used any supportive antioxidant, abused any drug, or had any metabolic disorder. None had any major disease or cancer except their kidney tumors. All patients were newly diagnosed, and their blood samples had been obtained in the preoperative period.

The 31 patients who made up the control group (21 males, 10 females) had a mean age of 45.4 ± 6.2. They were randomly selected from among volunteers who had no known major diseases and who did not use tobacco, alcohol, drugs, or antioxidants. Members of the patient and control groups had similar socioeconomic statuses.

According to the results of radiological and postoperative histopathological evaluation, 23 (69.7%) of the patients were in stage 1, 5 (15.2%) were in stage 2, and 5 (15.1%) were in stage 4 of metastatic renal cancer. The histopathological examinations revealed RCC with clear cell in 19 patients, chromophobe cell RCC in three patients, papillary types of RCCs in four patients, squamous cell carcinoma in two patients, unclassified RCC in one patient, sarcomatoid RCC in one patient, multilocular cystic RCC in one patient, and mixed-type RCC in two patients.

The study protocol was carried out in accordance with the Helsinki Declaration as revised in 1989. All participants were informed of the study protocol, and written consent was received from all participants.

Blood collection

Blood samples were obtained in the morning after 12 hours of fasting. Blood samples were collected in empty tubes and immediately stored on ice at 4 °C. Then, serum was separated from the cells by centrifugation at 1600g value for 10 minutes. Serum samples used to measure AD, CA, and CAT levels were stored at −20°C until they were used.

Measurement of AD activity

Serum AD activity was estimated spectrophotometrically using the Giusti method, which is based on the direct measurement of ammonia formation that occurs when AD is in the presence of excess adenosine.¹³ The results were expressed as units per liter (U/l).

Measurement of CA enzyme activity

Serum CA activity was assayed by CO₂ hydration. CO₂ hydration was measured using the Rickli and Wilbur-Anderson method, with bromothymol blue as the indicator.¹⁴ The results were expressed as U/l.

Measurement of CAT activity

Serum CAT activity was measured using H₂O₂ as a substrate.¹⁵ The degradation of H₂O₂ was monitored at 240 nm for 5 minutes using a spectrophotometer, and enzyme activity was expressed in U/l at 25 °C.

Statistical analysis

Descriptive statistics for the properties under study were expressed as mean, standard deviation, minimum value, and maximum value. A Student's t-test was used to compare the groups for these properties. To determine the relationships among them, the Pearson's correlation coefficient was calculated separately for each group. In addition, receiver operating characteristic analysis was conducted on both the patients and controls to find the cut-off value for the characteristics studied. The level of statistical significance was set at P < 0.05 for the calculations. SPSS Version 13 statistical software was used for the statistical analyses.

Results

Table 1 shows the demographic and clinical data of both the patient and control groups. There were no statistically significant differences between the groups with respect to age and body mass index (BMI) (P < 0.05) (Table 1).

Table 1 Demographic characteristics of the two groups

Parameters	Control (n = 31)	Patients (n = 33)	P-value
Age (years)	45.4 ± 6.2	54.1 ± 4.3	ns
BMI (kg/m^2)	23.56 ± 2.62	24.10 ± 1.33	ns
Gender	Female:10 Male:21	Female:9 Male:24	ns

Values are the mean ± SD; ns, non-significant.

AD, CA, and CAT activities were detected in all samples. Table 2 summarizes the mean activities of the enzymes. In the controls, the mean values of serum CAT and AD and CA activities were 25.4881, 7.165, and 0.1625 U/l, respectively (Table 2). In the patients, the mean values of activities of serum CAT, AD, and CA were 11.4701, 25.455, and 0.8368 U/l, respectively (Table 2).

Table 2 Serum AD, CA, and CAT activities in the two groups

	Control (n = 31)				Patients (n = 33)
Parameters	Median	Standard deviation	Min.	Max.	Median	Standard deviation	Min.	Max.	P-value
CAT	25.4881	2.54619	20.57	31.01	11.4701	11.44217	5.95	72.75	0.001
AD	7.165	1.3762	5.1	9.8	25.455	7.7976	15.0	37.2	0.001
CA	0.1625	0.05066	0.10	0.23	0.8368	0.14185	0.55	1.12	0.001

Both serum AD and CA activities were significantly higher in patients than in controls (all significant, P < 0.001). Serum CAT activities were significantly lower in patients than in controls (all significant, P < 0.001).

Discussion

In this study, we analyzed serum CA and CAT levels and the activity of serum AD, which is an important aminohydrolase in purine metabolism, in patients with RCC. In addition, we aimed to determine the relationship among the activities of these enzymes in RCC patients.

We observed that serum AD and CA activities were significantly higher in patients with RCC than in controls and that serum CAT activities were significantly lower in RCC patients than in controls. To the best of our knowledge, no other study has examined serum AD, CA, and CAT levels in patients with RCC.

Increased serum AD and CA and decreased CAT activities may play a role in the pathogenesis of RCC. Therefore, we believe that serum AD, CA, and CAT levels may be an informative prognostic marker for RCC. Knowing the levels of AD, CAT, and CA in RCC patients may be beneficial for planning postoperative treatment.

AD, which catalyzes the reaction in which adenosine is deaminated to inosine, has been accepted as an important enzyme in the maturation, proliferation, differentiation, and function of T lymphocytes.¹⁶ As an indicator of cellular immunity, the plasma activity of this enzyme has been suggested as being increased in diseases that cause a cell-mediated immune response, including cancer.¹⁷ Some studies have confirmed the value of estimating AD in the diagnosis and follow-up of patients with tuberculosis.¹⁸ Recent studies have focused on the diagnostic value of AD activity in typical pneumonia.¹⁹ Several studies have found increased AD activity in cancerous tissues and cells compared to noncancerous tissue.^8,9A study that examined AD and CAT activities both in tumor tissue and in normal bladder tissue found increased AD and decreased CAT activity in the cancerous tissue.⁹ However, a study of AD activities in the prostate tissues of prostate cancer patients and of controls found that AD levels were higher in the carcinomatous tissues than in the healthy ones.²⁰ Yet, other studies have found low AD activity in cancer patients.^10,11

There are few studies in the literature of AD activity in RCC cases. A study that examined the level of erythrocytes and lymphocytes AD in 31 RCC patients found decreased levels of lymphocytes AD, but it found decreased erythrocyte AD levels only in patients with blood types B and O. Their nephrectomy, lymphocytes, and erythrocytes AD levels were increased. Clinical progression of the disease has been found to be associated with decreased levels of lymphocytes AD in all patients and with increased levels only in patients with the blood group A.²¹

In the present study, we observed that serum AD activity was significantly higher in patients with RCC than in healthy controls. Increased AD activity might provide a selective advantage to cancer cells in growing and developing more rapidly. We speculate that increased AD activity might be a result of the leakage of the enzyme from primary tumor cells.

CAT, which is highly expressed in some tissues, protects cells against the excessive formation of reactive oxygen species and prevents the accumulation of H₂0₂ formed during oxygen transport. Although there is increasing evidence that CAT activity is suppressed in cancerous tissues,^9,22,23 some studies have found unchanged or increased activity in some tumor tissues.^24,25 Two studies observed that increased CAT activity in the tissues may occur to prevent the damage caused by oxidative stress in the cells of RCC patients.^26,27 In contrast, Durak et al.⁹ found decreased CAT activity in cancerous bladder tissues compared to both adjacent, cancer-free tissues and control bladder tissue.

Data from the present study clearly demonstrate significantly decreased CAT activity in the serum of RCC patients.

CAs are involved in several physiological and biological processes in humans. CA is not only a highly active enzyme but is also involved in cell–cell adhesion and cell proliferation²⁸ and plays various roles in various tissues. Species can produce many different CA isozymes, some of which act in the cytosol, while others are membrane bound. For example, in humans, there are three cytosolic isozymes (CA-I, II, and III), five membrane-bound isozymes, and several related proteins that lack catalytic activity. CA-I is found primarily in erythrocytes. The human CA-II isozyme is widely distributed and has been identified in the erythrocytes, brain, eyes, and kidneys.⁵ Carbonic anhydrase IX (CAIX) is a hypoxia-inducible member of the CA family that regulates intracellular pH, cell proliferation, cell adhesion, and tumor progression. Studies have demonstrated that high CAIX expression yields an aggressive tumor phenotype and a poor prognosis in many types of cancer. There is a significant relationship between CAIX protein and cell growth, even if it cannot clearly be demonstrated that this protein is a good indicator of tumor differentiation whose mechanism has not yet been clarified. CAIX has undertaken the active tasks and the use of biomarkers in regulating the cell cycle.

According to the results of two CA studies, increased expression of CAIX protein supports its use as a diagnostic marker in clear cell renal carcinoma²⁹ and colorectal tumors.³⁰ Another study found that the relationship between clear cell carcinoma of the kidney and CAIX membrane isoenzymes was more significant than previously reported. It is assumed that the cancer cell can be part of both the hypoxia-focused and the hypoxia-independent paths and that using this knowledge, a strategy to inhibit CAIX as part of managing malignancy may be developed.³¹

In the present study, we found significantly increased CA activity in the serum of RCC patients.

Conclusions

The current study is the first to investigate serum AD, CA, and CAT activities in patients with RCC. These markers might be important as an additional biochemical diagnostic tool for RCC. Further investigations using a larger cohort of RCC patients are needed to provide definitive data about the prognostic role of AD, CA, and CAT activities. We believe multidisciplinary studies are needed to aid the planning of patients’ preoperative and postoperative treatment and to design follow-up protocols.

Disclaimer statements

Contributors All authors contributed equally.

Funding None.

Conflicts of interest We hereby declare that this study has not been published or is under consideration for publication, in part or whole, elsewhere and that all authors have seen and approved the final version of the manuscript. All authors have made a significant contribution to the findings and methods in the paper. There are no financial or commercial interests.

Ethics approval The study is approved by the Ethics Commitee of Van yüzüncü Yıl University. The original Ethics Commitee Report with the registration number 14.02.2013/15.

ORCiD

Necip Pirinççi http://orcid.org/0000-0002-5494-0984

Kadir Ceylan http://orcid.org/0000-0002-9783-4831