Aging Clin Exp Res (2014) 26:7–12 DOI 10.1007/s40520-013-0176-9
ORIGINAL ARTICLE
Oxidative stress and aging: correlation with clinical parameters Ana Carla da Cruz • Fabricia Petronilho • Claudia Cipriano Vidal Heluany Francieli Vuolo • Samantha Pereira Miguel • Joa˜o Quevedo • Marco Aure´lio Romano-Silva • Felipe Dal-Pizzol
•
Received: 3 July 2012 / Accepted: 25 July 2013 / Published online: 3 December 2013 Ó Springer International Publishing Switzerland 2013
Abstract Background and aims The free radical theory of aging has been receiving a lot of attention in the past years. The aim of this study was to examine the correlation between oxidative damage, antioxidant enzyme activities and plasma antioxidant potential with clinical parameters in elderly people. Methods Elderly subjects over 80 years old were included in the study. Clinical data were collected based on the Cumulative Illness Rating Scale (n = 132). In addition, blood samples were collected to determine biochemical and oxidative stress. Results The results showed that the mean age of the participants was 85.1 ± 4.0 years old. Diabetic patients A. C. da Cruz F. Petronilho C. C. V. Heluany F. Vuolo F. Dal-Pizzol (&) Laborato´rio de Fisiopatologia Experimental and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil e-mail: [email protected] F. Petronilho S. P. Miguel Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Universidade do Sul de Santa Catarina, Tubara˜o, SC, Brazil J. Quevedo Laborato´rio de Neurocieˆncias and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina, Programa de Po´sGraduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil M. A. Romano-Silva Laborato´rio de Neurocieˆncias, Departamento de Sau´de Mental, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
presented higher plasma protein carbonyl levels when compared with non-diabetic, and plasma levels of thiobarbituric acid-reactive substances were correlated to serum triglyceride and LDL fraction. In contrast, a lower plasma total antioxidant capacity presented a relation with the presence of diabetes and arterial hypertension. In addition, healthy elderly subjects presented a higher plasma total antioxidant capacity. Conclusion Thus, it seemed that plasma antioxidant potential is a better predictor of successful aging in the elderly than oxidative damage parameters or plasma antioxidant enzyme activities. Keywords Elderly people Oxidative damage Antioxidant enzyme
Introduction In general, age is the consequence of two independent processes: progressive deterioration of physiological function and waste of resistance or adaptability to stress [1]. Many changes are associated with aging, such as an overall function decline, homeostasis decrease and increasing probability of acquiring degenerative diseases [2]. Aging is not a uniform phenomenon in humans; it is influenced by gender, race, social and economic conditions, demographic region, origin and location of the residence [3]. This determines the need to study the aging process, mainly in the old-aged people ([80 years) [4]. This portion of the population is the best example of successful aging since they have escaped major age-related diseases and have reached the extreme limit of human life. According to the available information, about 30–50 percent of
123
8
centenarians are in relatively good clinical condition, despite their advanced age. Thus, it can be concluded that they represent the optimal combination of an appropriate lifestyle and genetic background [5]. The free radical theory of aging has been receiving a lot of attention in the past years. According to the theory, reactive oxygen species, such as superoxide, hydrogen peroxide, hydroxyl radical, and reactive nitrogen species, such as nitric oxide, are constantly produced leading to oxidative damage. The cumulative damage during life span could be a major factor associated with the aging process [6], and this opens the perspective to perform antioxidant treatment of aging and age-related diseases; but to date, there are few studies that correlate, in humans, oxidative stress with old-aging [7]. In this study, we surveyed elderly people over 80 years old living in Sidero´polis, a city of about 12,000 inhabitants located in southern Santa Catarina, Brazil. A correlation was made between plasma oxidative stress parameters and clinical data.
Methods Sample population The sample consisted of all individuals over 80 years old living in Sidero´polis, Brazil. Data were transversally collected from May to November 2005, after the approval by the Ethics Committee of the Universidade do Extremo Sul Catarinense. The elderly people aged over 80 years in this city numbered 135 (source: registration of the Health Office of the City—Basic Health Care Information System—SIAB-2005). Study participants met the inclusion criteria (80 years old or older on January 1st, 2005 and reside in the city of Sidero´polis during the data collection period, year 2005) and agree to participate. The number of refusals to participate in this study was only three out of 135 individuals, and only two out of these 132 did not agree to blood collection. A structured interview was used for clinical data collection, and the general medical comorbidity was assessed by the Cumulative Illness Rating Scale (CIRS). Individuals in the lower 10 % percentile in the CIRS were grouped as healthy individuals to perform statistical analyses. Blood samples were collected after fasting and plasma/ serum was separated from the red cells through conventional centrifuging for routine clinical biochemistry tests: complete blood count (CBC) method, ABX automation, Pentra 60; glucose, diabetes, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, Triglyceride, VCM, folic acid, vitamin B12. In addition, plasma samples were immediately placed
123
Aging Clin Exp Res (2014) 26:7–12
on liquid nitrogen and stored at -80 °C for the determination of oxidative parameters. Thiobarbituric acid-reactive species As an index of oxidative stress, we used the formation of TBARS during an acid-heating reaction, as previously described [8]. In brief, the samples were mixed with 1 mL of 10 % trichloroacetic acid and 1 mL of 0.67 % thiobarbituric acid, and then heated in a boiling water bath for 15 min. TBARS was determined by the absorbance at 535 nm using 1,1,3,3-tetramethoxypropane as an external standard. To minimize peroxidation during the assay procedure, butylated hydroxytoluene was added to the TBA reagent mixture. Results were expressed as malondialdehyde (MDA) equivalents (nmol) per milligram of protein. Protein carbonyls The oxidative damage to proteins was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine, as previously described [9]. In brief, proteins were precipitated by the addition of 20 % trichloroacetic acid and redissolved in dinitrophenylhydrazine, and the absorbance was read at 370 nm. Results were expressed as protein carbonyls (nmol) per milligram of protein. Measurement of plasma superoxide dismutase and catalase activities Catalase (CAT) activity was measured by the rate of decrease in the hydrogen peroxide absorbance at 240 nm and expressed as U/mg of protein, as described elsewhere [10]. Superoxide dismutase (SOD) activity was assayed by measuring the inhibition of adrenaline auto-oxidation, as previously described [11]. Total antioxidant activity (FRAP assay) A modified method of Benzie and Strain [12], was adopted for the FRAP assay [13]. The stock solutions included 300 mM acetate buffer (3.1 g CH3COONa and 16 ml CH3OOH), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl36H2O solution. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ, and 2.5 ml FeCl36H2O. The temperature of the solution was raised to 37 °C before using. Plasma samples were allowed to react with the FRAP solution for 30 min in the dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593 nm. The standard curve was linear between 200 and 1,000 lM FeSO4. Results are expressed in lM Fe(II)/g and compared with that of ascorbic acid.
Aging Clin Exp Res (2014) 26:7–12
9
Statistical analysis All analyzed data were normally distributed as assessed by the Kolmogorov–Smirnov test. Demographic and clinical characteristics of the study groups were compared by t test
or ANOVA followed by Tukey post hoc test, as appropriate, and a two-sided significance level of 0.05 or less was considered statistically significant. Correlation between studied parameters was performed by Pearson’s correlation.
Table 1 Descriptive data of population studied Mean ± SD Age
N (%)
85 ± 4
Gender female
79 (60)
Hypertension
91 (69)
Ischemic heart disease
51 (39)
Diabetes mellitus
20 (16)
Stroke
14 (11)
Chronic obstructive pulmonary disease
14 (11)
Dementia
Table 2 Antioxidant defenses in older-aged individuals and their relationship with clinical data
7 (5)
Variable
Results The mean age of studied population was 85.1 ± 4.0 years old, with a minimum of 80 years old and maximum of 97 years old. Women represented 60.6 %, hypertensive 69 %, ischemic heart disease 39 %, diabetes mellitus 16 %, stroke and chronic obstructive pulmonary disease 11 % and dementia 5 % of the studied population (Table 1). Plasma antioxidant enzyme activities (CAT and SOD) did not correlate with any clinically relevant data in this
SOD activity (U/mg protein)
p
CAT activity (U/mg protein)
p
FRAP [lM Fe(II)/g]
p
Age 80–84
1.7 ± 1.2
85–89
1.2 ± 0.9
90–94
0.98 ± 0.76
1.5 ± 1.0
875.50 ± 432.45
1.3 ± 0.8
1.4 ± 1.1
775.12 ± 287.31
C95
0.912
1.6 ± 1.3
0.821
1.9 ± 1.2
767.50 ± 377.60
0.857
881.12 ± 534.10
Sex Male
1.4 ± 0.8
Female
1.3 ± 0.7
0.574
1.8 ± 0.9
0.546
1.7 ± 1.1
737.50 ± 434.80
0.657
689.42 ± 372.10
Total cholesterol \200 mg/dl [200 mg/dl HDL cholesterol
0.323 ± 1.67
0.227
1.74 ± 1.18
[55 mg/dl
0.172 ± 1.22
\55 mg/dl
1.70 ± 2.47
1.97 ± 2.92
0.06
3.10 ± 3.53 0.579
2.50 ± 3.16
787.50 ± 477.80
0.126
922.04 ± 571.42 0.515
3.08 ± 4.15
883.85 ± 545.04
0.143
706.10 ± 421.61
LDL cholesterol LDL \150 mg/dl
0.296 ± 1.60
LDL [150 mg/dl
1.69 ± 1.10
0.16
2.23 ± 3.06
0.245
2.94 ± 3.51
795.92 ± 489.39
0.213
925.82 ± 569.67
Triglycerides \200 mg/dl
0.180 ± 1.25
[200 mg/dl
2.04 ± 1.27
0.526
2.52 ± 3.27
0.531
3.03 ± 3.52
848.58 ± 520
0.341
914.23 ± 606.16
Hypertension Yes
0.116 ± 1.06
No
0.218 ± 1.28
0.655
2.7 ± 3.24
0.396
2.94 ± 3.41
778.86 ± 494.05
0.001
1,062.09 ± 586.97
Diabetes Differences between groups were determined by ANOVA (age) or t test (sex, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, hypertension, diabetes, CIRS)
Yes
1.41 ± 8.88
No CIRS
0.169 ± 1.21
Healthy Sick
0.317 ± 1.75 9.44 ± 0.84
0.582
2.90 ± 3.15
0.64
2.48 ± 3.2 0.364
3.01 ± 3.25 2.46 ± 3.39
332.88 ± 397.89
\0.001
951.14 ± 509.92 0.435
1,680.45 ± 333.38
\0.001
591.33 ± 237.06
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Table 3 Oxidative damage in older-aged individuals and its relationship with clinical data
Aging Clin Exp Res (2014) 26:7–12
Variable
Protein carbonyls (nmol/mg protein)
p
TBARS levels (nmol/mg protein)
p
80–84
2.43 ± 1.55
0.768
2.75 ± 1.21
0.753
85–89
3.31 ± 1.1
90–94
3.12 ± 1.3
2.25 ± 1.74
C95
3.68 ± 1.23
2.18 ± 1.71
Age 2.86 ± 1.89
Sex Male
2.12 ± 1.83
Female
2.57 ± 1.17
0.752
2.21 ± 1.13
0.631
2.65 ± 1.31
Total cholesterol \200 mg/dl [200 mg/dl HDL cholesterol
3.43 ± 1.75
0.699
3.31 ± 1.68
[55 mg/dl
3.42 ± 1.73
\55 mg/dl
3.04 ± 1.61
2.35 ± 1.23
0.083
2.69 ± 0.92 0.366
2.55 ± 1.09
0.558
2.39 ± 1.07
LDL cholesterol \150 mg/dl
3.41 ± 1.75
[150 mg/dl
3.32 ± 1.67
0.76
2.35 ± 1.18
0.05
2.72 ± 0.96
Triglycerides \200 mg/dl
3.32 ± 1.70
[200 mg/dl
3.60 ± 1.82
0.500
2.43 ± 1.04
0.01
3.07 ± 1.24
Hypertension Yes
3.34 ± 1.75
No
3.43 ± 1.62
0.781
2.56 ± 1.09
0.588
2.45 ± 1.09
Diabetes Differences between groups were determined by ANOVA (age) or t test (sex, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, hypertension, diabetes, CIRS)
Yes
4.29 ± 1.64
No CIRS
3.20 ± 1.67
Healthy
3.39 ± 1.70
Sick
3.35 ± 1.74
population (Table 2). Diabetic patients presented higher plasma protein carbonyl levels when compared with nondiabetic (Table 3), and plasma TBARS levels were correlated with serum triglyceride and LDL (Table 3). We could not find any other significant relationship between oxidative damage parameters and clinical data in the studied population. In contrast, lower plasma total antioxidant capacity presented a significant relation with the presence of diabetes and arterial hypertension (Table 2). In addition, healthy elderly (determined by the CIRS scale) presented a higher plasma total antioxidant capacity (Table 2).
Discussion The aging process includes changes that increase the risk of diseases and death, and this could be related to the accumulation of oxidative damage [14–19]. A chronic state of
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0.009
2.93 ± 1.23
0.07
2.46 ± 1.05 0.915
2.72 ± 1.24
0.341
2.50 ± 1.06
oxidative stress exists in cells of aerobic organisms even under normal physiological conditions, and this leads to a steady-state accumulation of oxidative damage in a variety of macromolecules during aging, resulting in a progressive loss in the functional efficiency of various cellular processes (the oxidative stress theory of aging). Beckman and Ames [20] made a useful addition to this debate by dividing the hypothesis into ‘‘strong’’ and ‘‘weak’’ versions. The strong version of the theory states that oxidative damage determines life span, while the weaker version postulates that oxidative damage is ‘‘associated’’ with age-related disease. It seems that the weak version of the oxidative theory of aging is already well established, but in this study we could not demonstrate a clear relationship between plasma oxidative damage parameters and the clinical performance of an old-aged population [20]. In contrast, Mezzetti et al. [21] showed that plasma peroxides are higher in elderly than in younger human subjects, and even higher in disabled
Aging Clin Exp Res (2014) 26:7–12
octo-nonagenarians, and lipid oxidation was higher in octogenarians with carotid atherosclerosis than in those with successful vascular aging [22]. In contrast, no apparent relationship was found between MDA levels and age in a study performed by Block et al. [23]. A Brazilian study demonstrated that plasma concentration of TBARS increased significantly in individuals of over 50 years of age, as compared to the 20–29 years group, but no relation to successful aging was demonstrated in this study [17]. Most of the published studies concerning aging and oxidative stress have mainly focused on the antioxidant component rather than in oxidized products derived from the action of reactive species on macromolecules. Longevity has been associated with higher a-tocopherol plasma content in mammalian species, as well as with serum carotenoids, and ascorbic acid. Healthy centenarians showed higher plasma levels of vitamin E and A than elderly subjects [24]. Similar results were reported by Paolisso in healthy centenarians from Italy, that showed higher plasma levels of vitamins C and E than aged subjects, and this was supported by the results on FRAP in our study [25]. Results showed by Skalska et al. [26] indicate that in hypertensive and diabetic patients, higher plasma endothelin-1 level was independently associated with lower plasma antioxidant status measured by FRAP and decreased vitamin C concentration, which may be a result of increased oxidative stress in these diseases thus further verifying in healthy elderly who presented higher plasma total antioxidant capacity. All these data indicated that unsuccessful aging implies a lower non-enzymatic antioxidant repertoire and that plasma antioxidants might predict aging conditions. Despite the fact that humans have higher tissue SOD concentration than short-living mammalian species, little information on antioxidant enzymes activity in human aging is available in the literature. Andersen et al. [27] found an age-related decrease in SOD and glutathione reductase activity, together with no changes in the activities of glutathione peroxidase and CAT with increasing age. In contrast, Macocci et al. [24] showed that in plasma and red blood cell, SOD activities tend to increase with increasing age. Any significant differences in plasma SOD and CAT in relation to the successful age could be found in this study.
Conclusion Findings in this study did not provide a correlation between successful aging and plasma oxidative damage parameters. It seemed that plasma antioxidant potential is a better predictor of successful aging in older elderly subjects, than oxidative damage parameters or plasma antioxidant enzyme activities.
11 Conflict of interest
None.
References 1. Kasapoglu M, Ozben T (2001) Alterations of antioxidant enzymes and oxidative stress markers in aging. Exp Gerontol 36:209–220 2. Barja G (2002) Endogenous oxidative stress relationship to aging, longevity and calorie restriction. Ageing Res Rev 1:397–411 3. Organizacio´n Panamericana de La Salud (2003) Guia Clı´nica para Atencio´n Primaria a las Personas Mayores, 3a edn. OPAS, Washington DC 4. Pluoffe LA (2003) Addressing social and gender inequalities in health among seniors in Canada. Cadernos de Saude Publica 19:855 5. Sikora E (2000) Studies on successful aging and longevity: Polish centenarian program. Acta Biochim Pol 47:487–489 6. Niki E (2000) Oxidative stress and aging. Intern Med 39:324–326 7. Golden RT, Hinerfeld AD, Melov S (2002) Oxidative stress and aging: beyond correlation. Aging Cell 1:117–123 8. Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431 9. Levine RL, Garland D, Oliver CN (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478 10. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126 11. Bannister JV, Calabrese L (1987) Assays for superoxide dismutase. Methods Biochem Anal 32:279–312 12. Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Anal Biochem 239:70–76 13. Rice-Evance C, Miller NJ (1994) Total antioxidant status in plasma and body fluids. Methods Enzymol 234:279–293 14. El Assar M, Angulo J, Vallejo S et al (2012) Mechanisms involved in the aging-induced vascular dysfunction. Front Physiol 3:132 15. Sanz A, Stefanatos RK (2008) The mitochondrial free radical theory of aging: a critical view. Curr Aging Sci 1:10–21 16. Cesari M, Kritchevsky SB, Nicklas B et al (2012) Oxidative damage, platelet activation, and inflammation to predict mobility disability and mortality in older persons: results from the health aging and body composition study. J Gerontol A Biol Sci Med Sci 67:671–676 17. Junqueira VB, Barros SB, Chan SS et al (2004) Aging and oxidative stress. Mol Aspects Med 25:5–16 18. Nakamura T, Cho DH, Lipton SA (2012) Redox regulation of protein misfolding, mitochondrial dysfunction, synaptic damage, and cell death in neurodegenerative diseases. Exp Neurol 238:12–21 19. Romano AD, Serviddio G, de Matthaeis A et al (2010) Oxidative stress and aging. J Nephrol 15:S29–S36 20. Beckman KB, Ames BN (1998) The free radical theory of aging matures. Physiology 78:547–581 21. Mezzetti A, Lafenna D, Romano F et al (1996) Systemic oxidative stress and its relationship with age and illness. J Am Geriatr Soc 44:823–827 22. Cherubini A, Zuliani G, Costantini F et al (2001) High vitamin E plasma levels and low density lipoprotein oxidation are associated with the absence of atherosclerosis in octogenarians. J Am Geriatr Soc 49:651–654 23. Block G, Dietrich M, Norkus EP, Packer L (2003) Oxidative stress in human populations. In: Cutler RG, Rodriguez H Critical reviews of oxidative stress and aging. Advances in basic science, diagnostics and intervention 2000, 2:870–880
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12 24. Mecocci P, Polidori MC, Troiano L et al (2000) Plasma antioxidants and longevity: a study on healthy centenarians. Free Radic Biol Med 28:1243–1248 25. Paolisso G, Tagliamonte MR, Rizzo MR et al (1998) Oxidative stress and advancing age: results in healthy centenarians. J Am Geriatr Soc 46:833–838
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Aging Clin Exp Res (2014) 26:7–12 26. Skalska AB, Pietrzycka A, Stepniewski M (2009) Correlation of endothelin 1 plasma levels with plasma antioxidant capacity in elderly patients treated for hypertension. Clin Biochem 42:358–364 27. Andersen HR, Nielsen JB, Nielsen F, Gransdjean P (1997) Antioxidative enzyme activities in human erythrocytes. Clin Chem 43:562–568
ORIGINAL ARTICLE
Oxidative stress and aging: correlation with clinical parameters Ana Carla da Cruz • Fabricia Petronilho • Claudia Cipriano Vidal Heluany Francieli Vuolo • Samantha Pereira Miguel • Joa˜o Quevedo • Marco Aure´lio Romano-Silva • Felipe Dal-Pizzol
•
Received: 3 July 2012 / Accepted: 25 July 2013 / Published online: 3 December 2013 Ó Springer International Publishing Switzerland 2013
Abstract Background and aims The free radical theory of aging has been receiving a lot of attention in the past years. The aim of this study was to examine the correlation between oxidative damage, antioxidant enzyme activities and plasma antioxidant potential with clinical parameters in elderly people. Methods Elderly subjects over 80 years old were included in the study. Clinical data were collected based on the Cumulative Illness Rating Scale (n = 132). In addition, blood samples were collected to determine biochemical and oxidative stress. Results The results showed that the mean age of the participants was 85.1 ± 4.0 years old. Diabetic patients A. C. da Cruz F. Petronilho C. C. V. Heluany F. Vuolo F. Dal-Pizzol (&) Laborato´rio de Fisiopatologia Experimental and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina, Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil e-mail: [email protected] F. Petronilho S. P. Miguel Programa de Po´s-Graduac¸a˜o em Cieˆncias da Sau´de, Universidade do Sul de Santa Catarina, Tubara˜o, SC, Brazil J. Quevedo Laborato´rio de Neurocieˆncias and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina, Programa de Po´sGraduac¸a˜o em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Criciu´ma, SC, Brazil M. A. Romano-Silva Laborato´rio de Neurocieˆncias, Departamento de Sau´de Mental, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
presented higher plasma protein carbonyl levels when compared with non-diabetic, and plasma levels of thiobarbituric acid-reactive substances were correlated to serum triglyceride and LDL fraction. In contrast, a lower plasma total antioxidant capacity presented a relation with the presence of diabetes and arterial hypertension. In addition, healthy elderly subjects presented a higher plasma total antioxidant capacity. Conclusion Thus, it seemed that plasma antioxidant potential is a better predictor of successful aging in the elderly than oxidative damage parameters or plasma antioxidant enzyme activities. Keywords Elderly people Oxidative damage Antioxidant enzyme
Introduction In general, age is the consequence of two independent processes: progressive deterioration of physiological function and waste of resistance or adaptability to stress [1]. Many changes are associated with aging, such as an overall function decline, homeostasis decrease and increasing probability of acquiring degenerative diseases [2]. Aging is not a uniform phenomenon in humans; it is influenced by gender, race, social and economic conditions, demographic region, origin and location of the residence [3]. This determines the need to study the aging process, mainly in the old-aged people ([80 years) [4]. This portion of the population is the best example of successful aging since they have escaped major age-related diseases and have reached the extreme limit of human life. According to the available information, about 30–50 percent of
123
8
centenarians are in relatively good clinical condition, despite their advanced age. Thus, it can be concluded that they represent the optimal combination of an appropriate lifestyle and genetic background [5]. The free radical theory of aging has been receiving a lot of attention in the past years. According to the theory, reactive oxygen species, such as superoxide, hydrogen peroxide, hydroxyl radical, and reactive nitrogen species, such as nitric oxide, are constantly produced leading to oxidative damage. The cumulative damage during life span could be a major factor associated with the aging process [6], and this opens the perspective to perform antioxidant treatment of aging and age-related diseases; but to date, there are few studies that correlate, in humans, oxidative stress with old-aging [7]. In this study, we surveyed elderly people over 80 years old living in Sidero´polis, a city of about 12,000 inhabitants located in southern Santa Catarina, Brazil. A correlation was made between plasma oxidative stress parameters and clinical data.
Methods Sample population The sample consisted of all individuals over 80 years old living in Sidero´polis, Brazil. Data were transversally collected from May to November 2005, after the approval by the Ethics Committee of the Universidade do Extremo Sul Catarinense. The elderly people aged over 80 years in this city numbered 135 (source: registration of the Health Office of the City—Basic Health Care Information System—SIAB-2005). Study participants met the inclusion criteria (80 years old or older on January 1st, 2005 and reside in the city of Sidero´polis during the data collection period, year 2005) and agree to participate. The number of refusals to participate in this study was only three out of 135 individuals, and only two out of these 132 did not agree to blood collection. A structured interview was used for clinical data collection, and the general medical comorbidity was assessed by the Cumulative Illness Rating Scale (CIRS). Individuals in the lower 10 % percentile in the CIRS were grouped as healthy individuals to perform statistical analyses. Blood samples were collected after fasting and plasma/ serum was separated from the red cells through conventional centrifuging for routine clinical biochemistry tests: complete blood count (CBC) method, ABX automation, Pentra 60; glucose, diabetes, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, Triglyceride, VCM, folic acid, vitamin B12. In addition, plasma samples were immediately placed
123
Aging Clin Exp Res (2014) 26:7–12
on liquid nitrogen and stored at -80 °C for the determination of oxidative parameters. Thiobarbituric acid-reactive species As an index of oxidative stress, we used the formation of TBARS during an acid-heating reaction, as previously described [8]. In brief, the samples were mixed with 1 mL of 10 % trichloroacetic acid and 1 mL of 0.67 % thiobarbituric acid, and then heated in a boiling water bath for 15 min. TBARS was determined by the absorbance at 535 nm using 1,1,3,3-tetramethoxypropane as an external standard. To minimize peroxidation during the assay procedure, butylated hydroxytoluene was added to the TBA reagent mixture. Results were expressed as malondialdehyde (MDA) equivalents (nmol) per milligram of protein. Protein carbonyls The oxidative damage to proteins was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine, as previously described [9]. In brief, proteins were precipitated by the addition of 20 % trichloroacetic acid and redissolved in dinitrophenylhydrazine, and the absorbance was read at 370 nm. Results were expressed as protein carbonyls (nmol) per milligram of protein. Measurement of plasma superoxide dismutase and catalase activities Catalase (CAT) activity was measured by the rate of decrease in the hydrogen peroxide absorbance at 240 nm and expressed as U/mg of protein, as described elsewhere [10]. Superoxide dismutase (SOD) activity was assayed by measuring the inhibition of adrenaline auto-oxidation, as previously described [11]. Total antioxidant activity (FRAP assay) A modified method of Benzie and Strain [12], was adopted for the FRAP assay [13]. The stock solutions included 300 mM acetate buffer (3.1 g CH3COONa and 16 ml CH3OOH), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl36H2O solution. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ, and 2.5 ml FeCl36H2O. The temperature of the solution was raised to 37 °C before using. Plasma samples were allowed to react with the FRAP solution for 30 min in the dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593 nm. The standard curve was linear between 200 and 1,000 lM FeSO4. Results are expressed in lM Fe(II)/g and compared with that of ascorbic acid.
Aging Clin Exp Res (2014) 26:7–12
9
Statistical analysis All analyzed data were normally distributed as assessed by the Kolmogorov–Smirnov test. Demographic and clinical characteristics of the study groups were compared by t test
or ANOVA followed by Tukey post hoc test, as appropriate, and a two-sided significance level of 0.05 or less was considered statistically significant. Correlation between studied parameters was performed by Pearson’s correlation.
Table 1 Descriptive data of population studied Mean ± SD Age
N (%)
85 ± 4
Gender female
79 (60)
Hypertension
91 (69)
Ischemic heart disease
51 (39)
Diabetes mellitus
20 (16)
Stroke
14 (11)
Chronic obstructive pulmonary disease
14 (11)
Dementia
Table 2 Antioxidant defenses in older-aged individuals and their relationship with clinical data
7 (5)
Variable
Results The mean age of studied population was 85.1 ± 4.0 years old, with a minimum of 80 years old and maximum of 97 years old. Women represented 60.6 %, hypertensive 69 %, ischemic heart disease 39 %, diabetes mellitus 16 %, stroke and chronic obstructive pulmonary disease 11 % and dementia 5 % of the studied population (Table 1). Plasma antioxidant enzyme activities (CAT and SOD) did not correlate with any clinically relevant data in this
SOD activity (U/mg protein)
p
CAT activity (U/mg protein)
p
FRAP [lM Fe(II)/g]
p
Age 80–84
1.7 ± 1.2
85–89
1.2 ± 0.9
90–94
0.98 ± 0.76
1.5 ± 1.0
875.50 ± 432.45
1.3 ± 0.8
1.4 ± 1.1
775.12 ± 287.31
C95
0.912
1.6 ± 1.3
0.821
1.9 ± 1.2
767.50 ± 377.60
0.857
881.12 ± 534.10
Sex Male
1.4 ± 0.8
Female
1.3 ± 0.7
0.574
1.8 ± 0.9
0.546
1.7 ± 1.1
737.50 ± 434.80
0.657
689.42 ± 372.10
Total cholesterol \200 mg/dl [200 mg/dl HDL cholesterol
0.323 ± 1.67
0.227
1.74 ± 1.18
[55 mg/dl
0.172 ± 1.22
\55 mg/dl
1.70 ± 2.47
1.97 ± 2.92
0.06
3.10 ± 3.53 0.579
2.50 ± 3.16
787.50 ± 477.80
0.126
922.04 ± 571.42 0.515
3.08 ± 4.15
883.85 ± 545.04
0.143
706.10 ± 421.61
LDL cholesterol LDL \150 mg/dl
0.296 ± 1.60
LDL [150 mg/dl
1.69 ± 1.10
0.16
2.23 ± 3.06
0.245
2.94 ± 3.51
795.92 ± 489.39
0.213
925.82 ± 569.67
Triglycerides \200 mg/dl
0.180 ± 1.25
[200 mg/dl
2.04 ± 1.27
0.526
2.52 ± 3.27
0.531
3.03 ± 3.52
848.58 ± 520
0.341
914.23 ± 606.16
Hypertension Yes
0.116 ± 1.06
No
0.218 ± 1.28
0.655
2.7 ± 3.24
0.396
2.94 ± 3.41
778.86 ± 494.05
0.001
1,062.09 ± 586.97
Diabetes Differences between groups were determined by ANOVA (age) or t test (sex, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, hypertension, diabetes, CIRS)
Yes
1.41 ± 8.88
No CIRS
0.169 ± 1.21
Healthy Sick
0.317 ± 1.75 9.44 ± 0.84
0.582
2.90 ± 3.15
0.64
2.48 ± 3.2 0.364
3.01 ± 3.25 2.46 ± 3.39
332.88 ± 397.89
\0.001
951.14 ± 509.92 0.435
1,680.45 ± 333.38
\0.001
591.33 ± 237.06
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Table 3 Oxidative damage in older-aged individuals and its relationship with clinical data
Aging Clin Exp Res (2014) 26:7–12
Variable
Protein carbonyls (nmol/mg protein)
p
TBARS levels (nmol/mg protein)
p
80–84
2.43 ± 1.55
0.768
2.75 ± 1.21
0.753
85–89
3.31 ± 1.1
90–94
3.12 ± 1.3
2.25 ± 1.74
C95
3.68 ± 1.23
2.18 ± 1.71
Age 2.86 ± 1.89
Sex Male
2.12 ± 1.83
Female
2.57 ± 1.17
0.752
2.21 ± 1.13
0.631
2.65 ± 1.31
Total cholesterol \200 mg/dl [200 mg/dl HDL cholesterol
3.43 ± 1.75
0.699
3.31 ± 1.68
[55 mg/dl
3.42 ± 1.73
\55 mg/dl
3.04 ± 1.61
2.35 ± 1.23
0.083
2.69 ± 0.92 0.366
2.55 ± 1.09
0.558
2.39 ± 1.07
LDL cholesterol \150 mg/dl
3.41 ± 1.75
[150 mg/dl
3.32 ± 1.67
0.76
2.35 ± 1.18
0.05
2.72 ± 0.96
Triglycerides \200 mg/dl
3.32 ± 1.70
[200 mg/dl
3.60 ± 1.82
0.500
2.43 ± 1.04
0.01
3.07 ± 1.24
Hypertension Yes
3.34 ± 1.75
No
3.43 ± 1.62
0.781
2.56 ± 1.09
0.588
2.45 ± 1.09
Diabetes Differences between groups were determined by ANOVA (age) or t test (sex, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, hypertension, diabetes, CIRS)
Yes
4.29 ± 1.64
No CIRS
3.20 ± 1.67
Healthy
3.39 ± 1.70
Sick
3.35 ± 1.74
population (Table 2). Diabetic patients presented higher plasma protein carbonyl levels when compared with nondiabetic (Table 3), and plasma TBARS levels were correlated with serum triglyceride and LDL (Table 3). We could not find any other significant relationship between oxidative damage parameters and clinical data in the studied population. In contrast, lower plasma total antioxidant capacity presented a significant relation with the presence of diabetes and arterial hypertension (Table 2). In addition, healthy elderly (determined by the CIRS scale) presented a higher plasma total antioxidant capacity (Table 2).
Discussion The aging process includes changes that increase the risk of diseases and death, and this could be related to the accumulation of oxidative damage [14–19]. A chronic state of
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0.009
2.93 ± 1.23
0.07
2.46 ± 1.05 0.915
2.72 ± 1.24
0.341
2.50 ± 1.06
oxidative stress exists in cells of aerobic organisms even under normal physiological conditions, and this leads to a steady-state accumulation of oxidative damage in a variety of macromolecules during aging, resulting in a progressive loss in the functional efficiency of various cellular processes (the oxidative stress theory of aging). Beckman and Ames [20] made a useful addition to this debate by dividing the hypothesis into ‘‘strong’’ and ‘‘weak’’ versions. The strong version of the theory states that oxidative damage determines life span, while the weaker version postulates that oxidative damage is ‘‘associated’’ with age-related disease. It seems that the weak version of the oxidative theory of aging is already well established, but in this study we could not demonstrate a clear relationship between plasma oxidative damage parameters and the clinical performance of an old-aged population [20]. In contrast, Mezzetti et al. [21] showed that plasma peroxides are higher in elderly than in younger human subjects, and even higher in disabled
Aging Clin Exp Res (2014) 26:7–12
octo-nonagenarians, and lipid oxidation was higher in octogenarians with carotid atherosclerosis than in those with successful vascular aging [22]. In contrast, no apparent relationship was found between MDA levels and age in a study performed by Block et al. [23]. A Brazilian study demonstrated that plasma concentration of TBARS increased significantly in individuals of over 50 years of age, as compared to the 20–29 years group, but no relation to successful aging was demonstrated in this study [17]. Most of the published studies concerning aging and oxidative stress have mainly focused on the antioxidant component rather than in oxidized products derived from the action of reactive species on macromolecules. Longevity has been associated with higher a-tocopherol plasma content in mammalian species, as well as with serum carotenoids, and ascorbic acid. Healthy centenarians showed higher plasma levels of vitamin E and A than elderly subjects [24]. Similar results were reported by Paolisso in healthy centenarians from Italy, that showed higher plasma levels of vitamins C and E than aged subjects, and this was supported by the results on FRAP in our study [25]. Results showed by Skalska et al. [26] indicate that in hypertensive and diabetic patients, higher plasma endothelin-1 level was independently associated with lower plasma antioxidant status measured by FRAP and decreased vitamin C concentration, which may be a result of increased oxidative stress in these diseases thus further verifying in healthy elderly who presented higher plasma total antioxidant capacity. All these data indicated that unsuccessful aging implies a lower non-enzymatic antioxidant repertoire and that plasma antioxidants might predict aging conditions. Despite the fact that humans have higher tissue SOD concentration than short-living mammalian species, little information on antioxidant enzymes activity in human aging is available in the literature. Andersen et al. [27] found an age-related decrease in SOD and glutathione reductase activity, together with no changes in the activities of glutathione peroxidase and CAT with increasing age. In contrast, Macocci et al. [24] showed that in plasma and red blood cell, SOD activities tend to increase with increasing age. Any significant differences in plasma SOD and CAT in relation to the successful age could be found in this study.
Conclusion Findings in this study did not provide a correlation between successful aging and plasma oxidative damage parameters. It seemed that plasma antioxidant potential is a better predictor of successful aging in older elderly subjects, than oxidative damage parameters or plasma antioxidant enzyme activities.
11 Conflict of interest
None.
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