Age and Gender Related Changes in Total Antioxidant Response and Oxidative Stress in Patients with SchizophreniaCorrespondence Address :
Dr. Uma Devi P, e-mail: firstname.lastname@example.org
This study aims to measure total antioxidant response (TAR) and total peroxides (TP) in schizophrenia patients using the ferric reducing activity of plasma (FRAP), and to evaluate their relationship with oxidative stress. The effect of age, gender and severity on the levels of these total antioxidants-oxidants in the selected subjects were analyzed. We measured the plasma total antioxidant potential and total peroxides in 60 schizophrenia patients and in 60 well-matched non-schizophrenic control subjects. Oxidative stress has been implicated to play a major role in aging. Total antioxidant capacity/total peroxide of the clinical samples was measured using the latest spectrophotometric measurement method. Results showed that Plasma TAR was found to be lower in patients with schizophrenia than in those of the control group. On the contrary, the patients had high total plasma peroxide levels. Oxidative stress index (OSI) values of the patients were significantly higher than those of controls (P < 0.001). Total peroxide levels were found significantly higher (p < 0.01) in the elderly subjects, as compared to the young and adult population; in addition, TAR was higher in young and adults, than in the elderly people (p < 0.001). We also found more oxidative stress in schizophrenic males than in schizophrenic females (P > 0.01). This study indicates that schizophrenia is associated with increased oxidative stress and depleted antioxidant status in elderly people, and the clinical utility of antioxidants needs to be further evaluated in schizophrenia patients.
Schizophrenia, Antioxidant defense system, Total antioxidant status, Total peroxides, Oxidative Stress Index.
Free radicals and reactive oxygen species play a number of significant and diverse roles in neurodegenerative diseases, including schizophrenia. The brain and the nervous system are particularly prone to free radical damage. As the membrane lipids are very rich in polyunsaturated fatty acids and areas of the human brain are very rich in iron, these factors play an essential role in generating free radical species .The ability of a tissue or fluid to buffer the effects of reactive oxygen species is called total antioxidant capacity (1).
Free radicals adversely modify biologically active molecules and whole cells, and are implicated in a variety of complications and ageing (2), (3). Blood contains many antioxidant molecules that prevent and/or inhibit harmful free radical reactions (4). Plasma concentrations of antioxidants can be measured separately in the laboratory, but these measurements are time-consuming, labour intensive and costly. Since antioxidative effects of antioxidant components of plasma are additive, the measurement of total antioxidant potential (TAOP) reflects the antioxidative status of plasma. We evaluated the total antioxidative status of plasma with by FRAP assay, as proposed by Benzie IF, Strain JJ, 1996 (5); Benzie IF, Strain JJ, 1999 (6). The total antioxidant capacity (TAC) parameter summarizes the overall activity of non-enzymic antioxidants and antioxidant enzymes. It provides information about antioxidant types and their concentrations, without exact qualitative differentiation. We evaluated the total oxidative status of plasma by measuring the total peroxide level.
The free radical theory of aging, proposes that age-dependent deterioration in the cell function is related to the accumulation of molecular oxidative damage caused by reactive oxygen species. These ageâ€“associated alterations in the antioxidant status could also contribute to increased oxidative stress in aging. Several studies have explored the effect of aging on total antioxidant capacity of plasma and tissues. They showed reduced (7), (8), (9) or unchanged (10) antioxidant defence.
There is a large amount of convincing data demonstrating that reactive oxygen species (ROS) are involved in the initiation and development of many different forms of schizophrenia. The levels of oxidants and antioxidants in schizophrenia have been evaluated. However, measurements of total antioxidant response (TAR) and total peroxides in schizophrenia patients in different age groups and gender-based abnormalities are not evaluated up to date. Therefore, the objectives of this study were to investigate plasma TAR-TP levels in schizophrenia patients with different age groups and gender. Severity based abnormalities were also investigated.
The study was conducted in the Postgraduate and Research Department of Biochemistry, Dr.N.G.P.Arts and Science College, Coimbatore, during the month of September 2004 to June 2007. A total of 60 schizophrenic patients of age group 18-65 years of both sexes (30 males and 30 females), from good socio-economic backgrounds, were selected from Udhayam Mananala Kaapagam, a mental Health care centre, Coimbatore, Tamilnadu, India. They all met DSM-IV (Diagnostic and Statistical Manual of Mental Disorders-IV) criteria (American Psychiatric Association, 2000) (11) for schizophrenia. Schizophrenic subjects were divided into three groups: (1) Schizophrenia patients with age range between 15-30 years, (2) schizophrenics with age range between 31-45years, (2) schizophrenics in the age range of 46-65 years.
Sixty age and gender matched healthy normal control subjects with no individual and familial history of mental illness, were recruited to participate in this study. They included 30 males and 30 females. Their ages ranged from 15 to 65 years, with a mean age of (28.9Â±14.1) years. Both patients and controls were recruited during the same period, from Coimbatore district. Matching between the patients and controls was done according to sex and age. Study subjects were currently within normal ranges in their routine blood, urine, and faeces tests, electrocardiograph and radiographs; disorders associated with heart, brain, lung, liver, kidney and other pivotal organs were excluded.
The design and the layout of this project was carried out with the approval the Chairman, Kovai Medical Center and Hospitals, and due permission was obtained from the board of institutional review Committee of the Kongu mananala Arakkattalai, before the start of the work. Informed and written consent was obtained from all subjects prior to examination
Determination of plasma total antioxidant /Total Peroxide potential
Measurement of Total Antioxidant Response (TAR):
The TAC of the plasma was measured using a novel, automated, colorimetric measurement method developed by Benzie and Strain, and modified by Erel, 2004(12). In this method, hydroxyl radical, which is the most potent biological radical, is produced. In the assay, ferrous ion solution, which is present in Reagent 1, is mixed with hydrogen peroxide which is present in Reagent 2. The sequential produced radicals such as brown-colored dianisidine radical cation, produced by the hydroxyl radical, are also potent radicals. In this assay, the antioxidative effect of the sample against the potent free radicalâ€™s reactions, which is initiated by the produced hydroxyl radical, is measured. The assay results are expressed as mmol Trolox equivalent/L (6), (7), (12).The precision of this assay is excellent. Accurate measurements of TAC can be obtained in as little as 10 min, making this assay, eminently suitable for the clinical biochemistry laboratory (13).
Measurement of Total Peroxide Concentration (TP)
Total peroxide (TP) concentrations were determined using the FOX2 method (14), with minor modifications (13), (15). The FOX2 test system is based on the oxidation of ferrous ion to ferric ion by various types of peroxides contained within the plasma samples, to produce a colored ferric-xylenol orange complex whose absorbency can be measured. The FOX2 reagent was prepared by dissolving ammonium ferrous sulfate (9.8 mg) in 250 mM H2SO4 (10 mL), to give a final concentration of 250 μM ferrous ion in acid. This solution was then added to 90 ml of HPLC-grade methanol containing 79.2 mg butylated hydroxytoluene (BHT). Finally, 7.6 mg xylenol orange was added with stirring, to make the final working reagen
A total of 60 schizophrenia patients and 60 non-schizophrenia subjects were recruited. Table 1 illustrates the total antioxidant response of young, adult, and elderly schizophrenia subjects. The total antioxidant capacities were measured as FRAP assay, and total peroxides levels were measured by FOX2 method in the blood plasma of study and control subjects of different age groups.
In (Table/Fig 1) ,the comparison between the schizophrenia group and the control groups revealed that the level of TAR was significantly lower, and TP and OSI levels were significantly higher in all the study groups (p<0.01), but these changes were statistically more significant in the study group with age above 45 i-e elderly patients (p<0.001). There was also a significant decrease in the total antioxidant potential values for schizophrenia patients with age range above 45 (elderly), in comparison with schizophrenia patients with young and adult age groups, as well in elderly control subjects.
There was a significant difference in the levels of TAR, TP, and OSI of male and female study and control subjects. (Table/Fig 2). Results showed that male patients have more oxidative stress than female patients. Results showed that chronic schizophrenic patients have more oxidative damage than that of acute patients.
In the present study, we found that the oxidative/ antioxidative balance shifted towards oxidative status, namely increased oxidative stress was present in elderly patients with schizophrenia, as compared to healthy control subjects.
It was shown that ROS and other oxidants could be also formed in the normal physiological process (17). Increased ROS, in turn, enhance LPO products, and thus, lead to tissue injury. H2O2 and other derivatives of peroxides increase in some conditions, and diffuse into plasma. Here, antioxidant components of plasma overwhelm them, and they are simultaneously consumed (4).
When TP is measured, it means that the sum of many peroxides like protein peroxide, lipid peroxide and H2O2, are measured. Although it is known that H2O2 and lipid peroxides increase in schizophrenia (1), (9), (10), (18),(19), oxidative stress has not been evaluated through TP-TAR in schizophrenia. It has been reported by Bilal et al (2002) (7), that TAR level decreases in schizophrenia patients, and decreased plasma total antioxidant levels may be related to the progression of illness. It was shown in the present study, that TAR level also decreased in schizophrenia patients, as compared to control subjects.
The possible reasons for this increase in TP, might be the inevitable increase in lipid peroxides and ROS, including H2O2 in schizophrenia. Many antioxidant molecules found in blood, prevent or inhibit the harmful effects of free radicals (4). Whenever there is a decrease in antioxidants and/or an increase in oxidants, oxidant/antioxidant balance is impaired in favour of oxidants, and this is known as oxidative stress (20), (21). It is known that oxidative stress is responsible for tissue injury in many diseases, and contributes to the development of schizophrenia.
The total antioxidant capacity of blood plasma was found to depend on the age of the donors, with older donors showing lower TAC values (Table1). Relative contribution of the total antioxidant capacity to age â€“ related oxidative stress in schizophrenia, is not well defined so far. While some studies demonstrated that the antioxidant capacity of plasma decreases (9), others showed that it is unchanged (10). Our data supports the view that aging is associated with a loss of antioxidant capacity of plasma. Previous studies have demonstrated low antioxidant capacity in schizophrenia (18), (19), however, effect of aging was observed by few only. Our results are consistent with the results of Yao et al (2001), (22) who hypothesized that abnormal age â€“ related changes of plasma antioxidant proteins were seen in schizophrenia. Age related decrease in the antioxidant status of the plasma observed in our study, is consistent with the previous report showing the depletion of main antioxidants: vitamin C, alpha tocopherol, vitamin A, reduced glutathione (20) and decreased activities of antioxidant enzymes (23), (24) in schizophrenia, and provides the indication that this decrease may be involved in the mechanisms of free radical-induced damage to lipids, proteins, and DNA during aging.
Bilal et al (2002) (7) have reported a decrease in TAR levels in schizophrenia patients. Similarly, TAR level was found to be low in the study groups, in the present study. In the light of these data, a possible reason for the decrease in TAR in schizophrenia may be the decrease in other antioxidants like enzymatic and non enzymatic antioxida
Based on the data presented, free radicals and impaired antioxidants play an important role in the functioning of the brain and schizophrenia in elderly patients, and male schizophrenia patients have more oxidative stress as compared to female patients and those belonging to the young and adult category. Until now, we have not found the univocal molecular basis of schizophrenia, which might be due to free radicals. In future, we think that this argument should be confirmed by controlled, multi-centered, prospective studies, which shall confirm the exact mechanisms of oxidative stress in patients with different age groups of schizophrenia.
The authors are grateful to Dr. Nalla. G. Palanisamy, M.B.B.S., MD, the Chairman, and Dr. Thavamani D.Palanisamy, M.B.B.S., DCH, the secretary, Kovai medical center and hospitals, and Dr. N.G.P. Arts and Science College, Coimbatore, India, for providing encouragement and technical support to carryout this project.
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