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The influence of the serum vitamin C levels on oxidative stress biomarkers in elderly women
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Clinical Biochemistry 40 (2007) 1367 – 1372
The influence of the serum vitamin C levels on oxidative stress biomarkers in elderly women Clóvis Paniz a,b , André Bairros b , Juliana Valentini b,c , Mariele Charão b , Rachel Bulcão b,c , Angela Moro b , Tilman Grune d , Solange Cristina Garcia b,⁎ a
c
Post-Graduate Program on Biochemical Toxicology, Center of Natural and Exact Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil b Laboratory of Toxicology (LATOX), Department of Clinical and Toxicology Analysis, Center of Health Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil Post-Graduate Program on Pharmaceutical Sciences, Center of Health Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil d Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstraβe, Stuttgart, Germany Received 16 June 2007; received in revised form 13 August 2007; accepted 15 August 2007 Available online 29 August 2007
Abstract Objectives: To verify if there is influence of the vitamin C blood levels on oxidative stress markers in elderly people. In order to verify it, women from a public retirement home were compared to non-institutionalized ones; all of them were in healthy conditions. Design and methods: Vitamin C, albumin, reduced glutathione, malondialdehyde (MDA), protein carbonyls and δ-aminolevulinate dehydratase activity (ALA-D) were analyzed in older women either from a public retirement home (n = 45) or non-institutionalized (n = 22). Results: The institutionalized ones showed significant decrease for vitamin C levels (p = 0.002), ALA-D and MDA (p b 0.05). Correlations were found between vitamin C and both albumin and ALA-D, also between ALA-D and both protein carbonyls and age. Conclusions: The institutionalized women presented decreased vitamin C, albumin, MDA and ALA-D compared to non-institutionalized. Thus, it could be suggested that vitamin C tends to protect blood thiolic proteins. Moreover, its blood δ-aminoevulinate dehydratase activity seemed to be an additional biomarker of oxidation stress in healthy elderly. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Healthy elderly; Vitamin C; MDA; ALA-D activity; Institutionalized; Protein carbonyl; Oxidative stress; Aging
Introduction The aging population is a global concern and has been the root of much discussion in the last decade. The estimation, considering the worldwide population, is that the number of people with 60 years old or more will increase by more than 300% in the next 50 years, from 609 million in the year 2000 to almost two billion in 2050 [1]. In South America, and more specifically in Brazil, the increase in the elderly population, between 2000 and 2050, will ⁎ Corresponding author. Universidade Federal de Santa Maria, Campus Universitário, Caixa Postal 5061, CEP 97110-970, Santa Maria (RS), Brazil. Fax: +55 55 3220 8018. E-mail address: [email protected] (S.C. Garcia).
be greater than 400% [2]. In this way, aging has become an important social issue and public health concern [1]. In the elderly, the occurrence of chronic diseases, such as neurological and cardiovascular diseases, is one of the main causes in the development of incapacity associated with the aging process [3]. Besides, there are evidences indicating oxidative stress as an important factor in the development of the human aging process and age-associated pathological changes, especially chronic ones [4–6]. The imbalance between reactive oxygen species (ROS) production and antioxidant defenses determines the degree of oxidative stress [4]. When there is an increase in ROS production or a decrease of antioxidant defenses, this systemic antioxidant/ pro-oxidant imbalance will lead to the accumulation of oxidative damage, which in turn may lead to a modification of cellular
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.08.013
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proteins, lipids and DNA, reduce functional capacity and increase the risk of disease [4,7]. However, to protect the human organism from oxidative damage caused by free radicals and reactive species, the organism possesses an elaborate antioxidant defense system consisting of enzymes such as catalase, superoxide dismutase, glutathione peroxidase and numerous non-enzymatic antioxidants, including glutathione, vitamins A, C and E, ubiquinone and flavonoids [8]. Although there are evidences of the effects of oxidative stress on the aging process, there are few studies that evaluate if exogenous antioxidants such as vitamin C may have influence on oxidative stress biomarkers levels in healthy elderly people. In the same way, there are particularly few reports about oxidative stress status in elderly institutionalized in public retirement home, that constitute generally, an isolated group of its familiar relations, homes and friends. The aging process presents changes in the lifestyle, including many cases of physical, psychological and social loss [9] beyond economic difficulties, that possibly becomes this group more inclined to nutritional lacks and consequently lower defense against oxidative stress. For these reasons, in this study, we measured levels of serum vitamin C, serum albumin and reduced glutathione (GSH) levels in red blood cells. Oxidant biomarkers in lipids, such as malondialdehyde (MDA), in protein such as protein carbonyls (PCO) and also a possible marker, the δ-aminolevulinate dehydratase activity (ALA-D) were analyzed. The elderly institutionalized women were compared with the non-institutionalized women to verify if there are differences between the groups. Finally, were verified the possible correlations of the vitamin C levels and oxidative stress biomarkers in the healthy elderly women.
the blood was centrifuged at 1500×g for 10 min at 4 °C, the plasma was used to determine MDA and the erythrocytes were used for GSH measurement. For ALA-D activity, whole blood was used. For vitamins C, albumin and PCO, the blood was collected without anticoagulant and centrifuged at 1500×g for 10 min at room temperature. The serum was separately stored in microtubes and kept at −20 °C until analysis. Plasma MDA, serum vitamin C, RBC GSH and ALA-D were all processed immediately. Plasmatic vitamin C quantification Vitamin C analysis followed the method described by Lloyd et al., with modifications [10]. The serum samples were deproteinized with trichloroacetic acid (TCA) 15%, using 400 μL of sample and 800 μL of TCA 15%, vortex-mixed for 15 s and centrifuged at 1800×g for 15 min. Then, 400 μL of supernatant was removed to the other tube and 120 μL of DTC (solution of 5 mL of 2.4 dinitrophenylhydrazine 0.1 mol/L in H2SO4 4.5 M; 0.25 mL of thiourea 0.66 mol/L; 0.25 mL of cupric sulfate 0.027 mol/L) was added. Samples were vortex-mixed for 10 s, closed with filmed paper and incubated at 60 °C for 60 min. After, samples were transferred to an ice bath for 10 min, and then, 600 μL of H2SO4 12 M was added. Samples were vortex-mixed for 10 s and read at 520 nm. All samples were measurement in duplicate. It was used calibration curves with L(+)-ascorbic acid (Vetec Química Fina Ltda) to determine the concentration, following the same procedure of the samples. Serum albumin
Methods
The serum albumin was determined by spectrophotometry by standard methods with commercial kits using Automatic Chemical Analyzer, model Hitachi 917® (Roche Diagnostic Systems, Inc, Branchburg, NJ).
Group selection
Reduced glutathione quantification in erythrocytes
A total of 45 women institutionalized in public retirement home (mean 71 ± 6 years) and 22 non-institutionalized women (68 ± 6 years) participated in this study. All were from Santa Maria, Brazil. Patients with congenital neurological or psychiatric pathologies were excluded, as well as those that presented serious neurological or psychiatric pathologies, metabolic diseases, acute or serious chronic diseases. None of the subjects studied was taking antioxidant supplementations (vitamins or minerals) and all participants were non-smokers. Moreover, all elderly women were healthy. The study was approved by the committee of ethics in the research of the Federal University of Santa Maria-RS and informed consent was required from all participants, according to the guidelines of the local ethics committee (131/06).
The levels of reduced glutathione in RBC were measured by high performance liquid chromatography (HPLC), a method development in our laboratory [11]. The chromatographic equipment consisted of a gradient chromatography system Knauer® apparatus, WellChrom model, equipped with a quaternary pump, reservoir for solvents, dynamic mixer, an online vacuum solvent degasser with four canals, manual injector with loop of 20 μL and UV–Vis detector. Chromatographic control, data collection and processing were carried out using EUROCHROM 2000 SOFTWARE®, basic edition, 2.05 for Windows. The separation was achieved using a C18 reverse-phase column Eurospher-100 150 × 4 mm with 5 μm particle size and a guard column Eurospher-100 5 × 4 mm with 5 μm particle size. The mobile phase was a mixture of KH2PO4 (pH 3.8; 25 mmol/L) and methanol with gradient elution. The absorbance of the eluent was monitored at 330 nm and the total run time was 25 min. The column was thermostated at 40 °C in a thermostatization system for chromatographic columns (Chromacon®).
Sample collection From all subjects, 10 mL of blood sample was collected with and without anticoagulant and stored on ice. After the collections,
C. Paniz et al. / Clinical Biochemistry 40 (2007) 1367–1372
After centrifugation of blood, the erythrocytes had been separated and 100 μL was added to 40 μL of EDTA solution. Then, the erythrocytes were hemolysed with 100 μL of Triton X-100 20% and after this deproteinized with 100 μL of TCA 15%. The samples were kept in rest for 20 min and centrifuged for 30 min. After, 130 μL of acid supernatant was added to 500 μL of Tris–HCl buffer, pH 8.9, and 350 μL of 5.5′-Dithiobis (2-nitrobenzoic acid) 10 mM and 100 μL of phosphoric acid. After these procedures, the tubes were centrifuged for 10 min, and before the injection on chromatographic equipment each sample was filtered. The GSH levels in erythrocytes were calculated: GSH levels = GSH mM × hematocrite. Thus, the natural effect of the hematocrite was considered.
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incubation was carried out for 1 h a 37 °C. The reaction product was measured at 555 nm. Statistical analysis The analysis of the data was performed using software SPSS v. 8.0 for Windows (SPSS Inc., Chicago, IL). Student's t-test was used to check significant differences between groups with variables normally distributed; when not, a non-parametric Mann– Whitney U-test was performed. Correlation tests were performed by the Pearson or Spearman correlation coefficient following the distribution of the variables. Values of p b 0.05 were considered significant. All results were expressed as mean± standard error mean (SEM).
Plasmatic MDA levels Results Quantification of lipid peroxidation was assessed by measuring MDA levels by HPLC with Vis detection as described by Grotto et al. [12]. This method analyzes the MDA levels after alkaline hydrolysis. Serum carbonylation of proteins The carbonylation of serum proteins was determined by modification of the Levine's method [13]. The serum total protein was determined by using commercial kits (Labtest Diagnóstica S.A.) and adjusted to 10 mg/mL with distilled/deionized water. After, 500 μL of serum solution was mixed with 250 μL of 10% trichloracetic acid (TCA), centrifuged at 1500×g for 5 min, and the supernatant was discarded. Following, 250 μL of 2.4dinitrophenylhydrazine (10 mmol/L in 2 mol/L HCl) was added to this precipitated protein, vortex-mixed until being homogeneous and incubated at room temperature for 30 min. During the incubation time the samples were mixed vigorously every 15 min. After the incubation time, 0.5 mL of 10% TCA was added to the protein precipitated and centrifuged at 1500×g for 5 min. After discarding the supernatant, precipitate was washed twice with 1 mL of ethanol/ethylacetate (1:1), each time centrifuging out the supernatant in order to remove the free DNPH. The precipitate was dissolved in 1.5 mL of protein dissolving solution (2 g SDS and 50 mg EDTA in 100 mL 80 mmol/L phosphate buffer, pH 8.0) and incubated at 37 °C for 10 min. The color intensity of the supernatant was measured using spectrophotometer at 370 nm against 2 mol/L HCl. Carbonyl content was calculated by using molar extinction coefficient (21 × 103 1/mol cm) and results were expressed as nanomoles per milligram protein. δ-Aminolevulinate dehydratase activity The δ-aminolevulinate dehydratase activity was assayed in the total blood by the Sassa's method [14] with minor modifications by measuring the rate of phorphobilinogen (PBG) formation in 1 h at 37 °C. The enzyme reaction was initiated after 10 min of pre-incubation. The reaction was started by adding δ-aminulevulinic acid (ALA) to a final concentration of 4 mM in phosphate buffered solution at pH 6.8, and
The results for vitamin C are shown in Table 1. The reference values for vitamin C, considering the dinitrophenylhydrazine method, vary from 23 to 85 μmol/L for adults [15]. Although they were within the normal range for adults, serum vitamin C levels were significantly decreased in the institutionalized individuals when compared to the non-institutionalized ones (p = 0.002). In the same way, the serum albumin also presented levels within the normal range, but it was significantly decreased in the institutionalized group (p b 0.01), as shown in Table 1. For GSH levels and PCO content, the groups did not present statistically significant differences (Table 1). On the other hand, plasmatic MDA levels and ALA-D activity were significantly decreased in the institutionalized ones (p = 0.029 and p = 0.001, respectively) when compared to the non-institutionalized group (Table 1). Another verified aspect was the possible correlation among the parameters analyzed. A positive correlation was found between vitamin C and albumin (Fig. 1) and between vitamin C and ALA-D activity (Fig. 2); and a negative correlation between ALA-D and PCO (Fig. 3) and ALA-D and age (Fig. 4). Discussion Previous studies have shown that vitamin C protects watersoluble components of the body. There are evidences that this antioxidant interacts in vivo in the aqueous phase with vitamin E [16], sparing or recycling lipid-bound vitamin E previously oxidized [17]. Table 1 Results of vitamin C, GSH, PCO, MDA, and ALA-D in blood of elder women, represented as mean values ± SEM Parameter
Institutionalized n = 45
Non-institutionalized n = 22
Vitamin C (μmol/L) Albumin (g/dL) GSH (mM) PCO (nmol/L) MDA (μM) ALA-D (U/L− 1)
53.86 ± 3.90⁎ 3.86 ± 0.05⁎ 0.75 ± 0.02 0.75 ± 0.03 4.43 ± 0.12⁎⁎ 11.32 ± 0.48⁎⁎
75.69 ± 6.22 4.24 ± 0.05 0.76 ± 0.04 0.73 ± 0.06 4.89 ± 0.17 14.40 ± 0.94
⁎p b 0.001; ⁎⁎p b 0.05.
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Fig. 1. Pearson correlations between vitamin C levels vs albumin in elder women (n = 67).
Fig. 3. Pearson correlation between ALA-D activity vs PCO in elder women (n = 67).
All the elderly women presented serum vitamin C levels within the normal range established for adults [15]. However, the levels of the institutionalized women were significantly decreased. In another study with elderly subjects (average age of 71.5 years), vitamin C levels were found to be 52.9 ± 3.0 [18]. These results were very similar to those found in this study. Nevertheless, the non-institutionalized group presented higher levels. The vitamin C is only obtained through the diet, thus these results could suggest inadequate nutritional intake in the group living in a public retirement home. The results of the correlations demonstrated that vitamin C levels were associated with blood protein levels, particularly, with albumin and ALA-D, which may be associated with the protection of thiol groups, once these groups are highly sensitive to pro-oxidant elements [19–21] that impair its function [22,23]. As albumin, via its thiol groups, is the main extracellular antioxidant molecule [24] and ALA-D is a thioldependent enzyme, they can represent better the circulating oxidative status. Vitamin C, in increased levels, probably plays
a protective role against the oxidation of -SH groups in these proteins. Thus, in elderly, higher levels of this nutrient could be necessary for an efficient protection against blood proteins-thiol oxidation verified in the non-institutionalized group due by water-soluble vitamin C characteristic. However, despite the inhibition of thiol groups in albumin, it could be decreased in institutionalized women also due to malnourishment from undetected chronic disease, as previously reported [25]. GSH in its reduced form is accepted as the most important and representative intracellular antioxidant. Reduced GSH is usually located in the red blood cells while its concentration is low in extracellular fluids [26]. On the other hand, MDA is a major product of the free radical attack on polyunsaturated fatty acids and it is widely used as a biomarker of lipid peroxidation [27] indicating the extent of cell membrane injury [28]. In addition, PCO is the most used biomarker for protein oxidative damage, and it reflects the cellular damage induced by multiple forms of ROS [29,30].
Fig. 2. Pearson correlations between vitamin C levels vs ALA-D activity in elder women (n = 67).
Fig. 4. Pearson correlation between ALA-D activity vs age in elder women (n = 67).
C. Paniz et al. / Clinical Biochemistry 40 (2007) 1367–1372
Considering these oxidative stress biomarkers, the differences in the erythrocyte GSH concentration and in serum PCO content were not significant between the groups. However, plasmatic MDA levels of the non-institutionalized group showed a significant increase in relation to the institutionalized ones. However, the MDA levels appear not to have relation with vitamin C levels, maybe because this vitamin is a water-soluble antioxidant. Moreover, other aspects that were not approached in this work also would be contributing to the lower levels of MDA in this group. On the other hand, ALA-D activity was significantly decreased in the institutionalized women. It is known that ALA-D is an enzyme of the heme biosynthesis pathway, essential for all aerobic organisms. It is a zinc metalloenzyme that requires reduced thiol groups for its activity [31]. ALA-D inhibition impairs heme biosynthesis and leads to the accumulation of its intermediates. One of them, δ-aminolevulinic acid (ALA), has been shown to induce pro-oxidant events [32,33]. An interesting and important finding of the present report was the fact that, at the first time in healthy elderly people, ALA-D activity was evaluated such as possible oxidative stress biomarkers. The results showed two interesting aspects: a positive correlation with an important plasma antioxidant, the vitamin C (Fig. 1), and a negative correlation with age and PCO (Fig. 2). The institutionalized and non-institutionalized groups did not present a significant age difference. However, a negative correlation between ALA-D and age was observed (Fig. 3). In opposition, GSH did not present a correlation with age (date not shown). Although it is known that GSH decreases with aging [34,35], this was not found in our study, probably due to the small age variation between both groups. Therefore, these results could suggest that ALA-D may be used to evaluate the relationship between -SH oxidation and aging, but other studies are necessary. Through the correlation with PCO, it was shown that this enzyme could be utilized as an additional protein oxidation marker. In conclusion, the exogenous antioxidant vitamin C presented levels within the normal range for adults. Institutionalized women showed vitamin C, albumin, MDA and ALA-D decrease compared to the non-institutionalized group. Considering the results, we suggest that vitamin C plays a possible protective role, probably avoiding thiolic group oxidation in plasma proteins. Moreover, ALA-D activity was presented as an oxidative stress biomarker in elderly people.
[3]
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[22]
Acknowledgments [23]
Work supported by CNPq (grant 402243/2005-6 to S.C. Garcia) and DAAD (grant to S.C. Garcia). M. Charão and A. Moro are recipients of the PIBIC Fellowships, respectively. S.C. Garcia is the recipient of CNPq Research Fellowship.
[24]
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Clinical Biochemistry 40 (2007) 1367 – 1372
The influence of the serum vitamin C levels on oxidative stress biomarkers in elderly women Clóvis Paniz a,b , André Bairros b , Juliana Valentini b,c , Mariele Charão b , Rachel Bulcão b,c , Angela Moro b , Tilman Grune d , Solange Cristina Garcia b,⁎ a
c
Post-Graduate Program on Biochemical Toxicology, Center of Natural and Exact Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil b Laboratory of Toxicology (LATOX), Department of Clinical and Toxicology Analysis, Center of Health Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil Post-Graduate Program on Pharmaceutical Sciences, Center of Health Sciences, Federal University of Santa Maria, 97105-900, Santa Maria, RS, Brazil d Institute of Biological Chemistry and Nutrition, University of Hohenheim, Garbenstraβe, Stuttgart, Germany Received 16 June 2007; received in revised form 13 August 2007; accepted 15 August 2007 Available online 29 August 2007
Abstract Objectives: To verify if there is influence of the vitamin C blood levels on oxidative stress markers in elderly people. In order to verify it, women from a public retirement home were compared to non-institutionalized ones; all of them were in healthy conditions. Design and methods: Vitamin C, albumin, reduced glutathione, malondialdehyde (MDA), protein carbonyls and δ-aminolevulinate dehydratase activity (ALA-D) were analyzed in older women either from a public retirement home (n = 45) or non-institutionalized (n = 22). Results: The institutionalized ones showed significant decrease for vitamin C levels (p = 0.002), ALA-D and MDA (p b 0.05). Correlations were found between vitamin C and both albumin and ALA-D, also between ALA-D and both protein carbonyls and age. Conclusions: The institutionalized women presented decreased vitamin C, albumin, MDA and ALA-D compared to non-institutionalized. Thus, it could be suggested that vitamin C tends to protect blood thiolic proteins. Moreover, its blood δ-aminoevulinate dehydratase activity seemed to be an additional biomarker of oxidation stress in healthy elderly. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Healthy elderly; Vitamin C; MDA; ALA-D activity; Institutionalized; Protein carbonyl; Oxidative stress; Aging
Introduction The aging population is a global concern and has been the root of much discussion in the last decade. The estimation, considering the worldwide population, is that the number of people with 60 years old or more will increase by more than 300% in the next 50 years, from 609 million in the year 2000 to almost two billion in 2050 [1]. In South America, and more specifically in Brazil, the increase in the elderly population, between 2000 and 2050, will ⁎ Corresponding author. Universidade Federal de Santa Maria, Campus Universitário, Caixa Postal 5061, CEP 97110-970, Santa Maria (RS), Brazil. Fax: +55 55 3220 8018. E-mail address: [email protected] (S.C. Garcia).
be greater than 400% [2]. In this way, aging has become an important social issue and public health concern [1]. In the elderly, the occurrence of chronic diseases, such as neurological and cardiovascular diseases, is one of the main causes in the development of incapacity associated with the aging process [3]. Besides, there are evidences indicating oxidative stress as an important factor in the development of the human aging process and age-associated pathological changes, especially chronic ones [4–6]. The imbalance between reactive oxygen species (ROS) production and antioxidant defenses determines the degree of oxidative stress [4]. When there is an increase in ROS production or a decrease of antioxidant defenses, this systemic antioxidant/ pro-oxidant imbalance will lead to the accumulation of oxidative damage, which in turn may lead to a modification of cellular
0009-9120/$ - see front matter © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2007.08.013
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proteins, lipids and DNA, reduce functional capacity and increase the risk of disease [4,7]. However, to protect the human organism from oxidative damage caused by free radicals and reactive species, the organism possesses an elaborate antioxidant defense system consisting of enzymes such as catalase, superoxide dismutase, glutathione peroxidase and numerous non-enzymatic antioxidants, including glutathione, vitamins A, C and E, ubiquinone and flavonoids [8]. Although there are evidences of the effects of oxidative stress on the aging process, there are few studies that evaluate if exogenous antioxidants such as vitamin C may have influence on oxidative stress biomarkers levels in healthy elderly people. In the same way, there are particularly few reports about oxidative stress status in elderly institutionalized in public retirement home, that constitute generally, an isolated group of its familiar relations, homes and friends. The aging process presents changes in the lifestyle, including many cases of physical, psychological and social loss [9] beyond economic difficulties, that possibly becomes this group more inclined to nutritional lacks and consequently lower defense against oxidative stress. For these reasons, in this study, we measured levels of serum vitamin C, serum albumin and reduced glutathione (GSH) levels in red blood cells. Oxidant biomarkers in lipids, such as malondialdehyde (MDA), in protein such as protein carbonyls (PCO) and also a possible marker, the δ-aminolevulinate dehydratase activity (ALA-D) were analyzed. The elderly institutionalized women were compared with the non-institutionalized women to verify if there are differences between the groups. Finally, were verified the possible correlations of the vitamin C levels and oxidative stress biomarkers in the healthy elderly women.
the blood was centrifuged at 1500×g for 10 min at 4 °C, the plasma was used to determine MDA and the erythrocytes were used for GSH measurement. For ALA-D activity, whole blood was used. For vitamins C, albumin and PCO, the blood was collected without anticoagulant and centrifuged at 1500×g for 10 min at room temperature. The serum was separately stored in microtubes and kept at −20 °C until analysis. Plasma MDA, serum vitamin C, RBC GSH and ALA-D were all processed immediately. Plasmatic vitamin C quantification Vitamin C analysis followed the method described by Lloyd et al., with modifications [10]. The serum samples were deproteinized with trichloroacetic acid (TCA) 15%, using 400 μL of sample and 800 μL of TCA 15%, vortex-mixed for 15 s and centrifuged at 1800×g for 15 min. Then, 400 μL of supernatant was removed to the other tube and 120 μL of DTC (solution of 5 mL of 2.4 dinitrophenylhydrazine 0.1 mol/L in H2SO4 4.5 M; 0.25 mL of thiourea 0.66 mol/L; 0.25 mL of cupric sulfate 0.027 mol/L) was added. Samples were vortex-mixed for 10 s, closed with filmed paper and incubated at 60 °C for 60 min. After, samples were transferred to an ice bath for 10 min, and then, 600 μL of H2SO4 12 M was added. Samples were vortex-mixed for 10 s and read at 520 nm. All samples were measurement in duplicate. It was used calibration curves with L(+)-ascorbic acid (Vetec Química Fina Ltda) to determine the concentration, following the same procedure of the samples. Serum albumin
Methods
The serum albumin was determined by spectrophotometry by standard methods with commercial kits using Automatic Chemical Analyzer, model Hitachi 917® (Roche Diagnostic Systems, Inc, Branchburg, NJ).
Group selection
Reduced glutathione quantification in erythrocytes
A total of 45 women institutionalized in public retirement home (mean 71 ± 6 years) and 22 non-institutionalized women (68 ± 6 years) participated in this study. All were from Santa Maria, Brazil. Patients with congenital neurological or psychiatric pathologies were excluded, as well as those that presented serious neurological or psychiatric pathologies, metabolic diseases, acute or serious chronic diseases. None of the subjects studied was taking antioxidant supplementations (vitamins or minerals) and all participants were non-smokers. Moreover, all elderly women were healthy. The study was approved by the committee of ethics in the research of the Federal University of Santa Maria-RS and informed consent was required from all participants, according to the guidelines of the local ethics committee (131/06).
The levels of reduced glutathione in RBC were measured by high performance liquid chromatography (HPLC), a method development in our laboratory [11]. The chromatographic equipment consisted of a gradient chromatography system Knauer® apparatus, WellChrom model, equipped with a quaternary pump, reservoir for solvents, dynamic mixer, an online vacuum solvent degasser with four canals, manual injector with loop of 20 μL and UV–Vis detector. Chromatographic control, data collection and processing were carried out using EUROCHROM 2000 SOFTWARE®, basic edition, 2.05 for Windows. The separation was achieved using a C18 reverse-phase column Eurospher-100 150 × 4 mm with 5 μm particle size and a guard column Eurospher-100 5 × 4 mm with 5 μm particle size. The mobile phase was a mixture of KH2PO4 (pH 3.8; 25 mmol/L) and methanol with gradient elution. The absorbance of the eluent was monitored at 330 nm and the total run time was 25 min. The column was thermostated at 40 °C in a thermostatization system for chromatographic columns (Chromacon®).
Sample collection From all subjects, 10 mL of blood sample was collected with and without anticoagulant and stored on ice. After the collections,
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After centrifugation of blood, the erythrocytes had been separated and 100 μL was added to 40 μL of EDTA solution. Then, the erythrocytes were hemolysed with 100 μL of Triton X-100 20% and after this deproteinized with 100 μL of TCA 15%. The samples were kept in rest for 20 min and centrifuged for 30 min. After, 130 μL of acid supernatant was added to 500 μL of Tris–HCl buffer, pH 8.9, and 350 μL of 5.5′-Dithiobis (2-nitrobenzoic acid) 10 mM and 100 μL of phosphoric acid. After these procedures, the tubes were centrifuged for 10 min, and before the injection on chromatographic equipment each sample was filtered. The GSH levels in erythrocytes were calculated: GSH levels = GSH mM × hematocrite. Thus, the natural effect of the hematocrite was considered.
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incubation was carried out for 1 h a 37 °C. The reaction product was measured at 555 nm. Statistical analysis The analysis of the data was performed using software SPSS v. 8.0 for Windows (SPSS Inc., Chicago, IL). Student's t-test was used to check significant differences between groups with variables normally distributed; when not, a non-parametric Mann– Whitney U-test was performed. Correlation tests were performed by the Pearson or Spearman correlation coefficient following the distribution of the variables. Values of p b 0.05 were considered significant. All results were expressed as mean± standard error mean (SEM).
Plasmatic MDA levels Results Quantification of lipid peroxidation was assessed by measuring MDA levels by HPLC with Vis detection as described by Grotto et al. [12]. This method analyzes the MDA levels after alkaline hydrolysis. Serum carbonylation of proteins The carbonylation of serum proteins was determined by modification of the Levine's method [13]. The serum total protein was determined by using commercial kits (Labtest Diagnóstica S.A.) and adjusted to 10 mg/mL with distilled/deionized water. After, 500 μL of serum solution was mixed with 250 μL of 10% trichloracetic acid (TCA), centrifuged at 1500×g for 5 min, and the supernatant was discarded. Following, 250 μL of 2.4dinitrophenylhydrazine (10 mmol/L in 2 mol/L HCl) was added to this precipitated protein, vortex-mixed until being homogeneous and incubated at room temperature for 30 min. During the incubation time the samples were mixed vigorously every 15 min. After the incubation time, 0.5 mL of 10% TCA was added to the protein precipitated and centrifuged at 1500×g for 5 min. After discarding the supernatant, precipitate was washed twice with 1 mL of ethanol/ethylacetate (1:1), each time centrifuging out the supernatant in order to remove the free DNPH. The precipitate was dissolved in 1.5 mL of protein dissolving solution (2 g SDS and 50 mg EDTA in 100 mL 80 mmol/L phosphate buffer, pH 8.0) and incubated at 37 °C for 10 min. The color intensity of the supernatant was measured using spectrophotometer at 370 nm against 2 mol/L HCl. Carbonyl content was calculated by using molar extinction coefficient (21 × 103 1/mol cm) and results were expressed as nanomoles per milligram protein. δ-Aminolevulinate dehydratase activity The δ-aminolevulinate dehydratase activity was assayed in the total blood by the Sassa's method [14] with minor modifications by measuring the rate of phorphobilinogen (PBG) formation in 1 h at 37 °C. The enzyme reaction was initiated after 10 min of pre-incubation. The reaction was started by adding δ-aminulevulinic acid (ALA) to a final concentration of 4 mM in phosphate buffered solution at pH 6.8, and
The results for vitamin C are shown in Table 1. The reference values for vitamin C, considering the dinitrophenylhydrazine method, vary from 23 to 85 μmol/L for adults [15]. Although they were within the normal range for adults, serum vitamin C levels were significantly decreased in the institutionalized individuals when compared to the non-institutionalized ones (p = 0.002). In the same way, the serum albumin also presented levels within the normal range, but it was significantly decreased in the institutionalized group (p b 0.01), as shown in Table 1. For GSH levels and PCO content, the groups did not present statistically significant differences (Table 1). On the other hand, plasmatic MDA levels and ALA-D activity were significantly decreased in the institutionalized ones (p = 0.029 and p = 0.001, respectively) when compared to the non-institutionalized group (Table 1). Another verified aspect was the possible correlation among the parameters analyzed. A positive correlation was found between vitamin C and albumin (Fig. 1) and between vitamin C and ALA-D activity (Fig. 2); and a negative correlation between ALA-D and PCO (Fig. 3) and ALA-D and age (Fig. 4). Discussion Previous studies have shown that vitamin C protects watersoluble components of the body. There are evidences that this antioxidant interacts in vivo in the aqueous phase with vitamin E [16], sparing or recycling lipid-bound vitamin E previously oxidized [17]. Table 1 Results of vitamin C, GSH, PCO, MDA, and ALA-D in blood of elder women, represented as mean values ± SEM Parameter
Institutionalized n = 45
Non-institutionalized n = 22
Vitamin C (μmol/L) Albumin (g/dL) GSH (mM) PCO (nmol/L) MDA (μM) ALA-D (U/L− 1)
53.86 ± 3.90⁎ 3.86 ± 0.05⁎ 0.75 ± 0.02 0.75 ± 0.03 4.43 ± 0.12⁎⁎ 11.32 ± 0.48⁎⁎
75.69 ± 6.22 4.24 ± 0.05 0.76 ± 0.04 0.73 ± 0.06 4.89 ± 0.17 14.40 ± 0.94
⁎p b 0.001; ⁎⁎p b 0.05.
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Fig. 1. Pearson correlations between vitamin C levels vs albumin in elder women (n = 67).
Fig. 3. Pearson correlation between ALA-D activity vs PCO in elder women (n = 67).
All the elderly women presented serum vitamin C levels within the normal range established for adults [15]. However, the levels of the institutionalized women were significantly decreased. In another study with elderly subjects (average age of 71.5 years), vitamin C levels were found to be 52.9 ± 3.0 [18]. These results were very similar to those found in this study. Nevertheless, the non-institutionalized group presented higher levels. The vitamin C is only obtained through the diet, thus these results could suggest inadequate nutritional intake in the group living in a public retirement home. The results of the correlations demonstrated that vitamin C levels were associated with blood protein levels, particularly, with albumin and ALA-D, which may be associated with the protection of thiol groups, once these groups are highly sensitive to pro-oxidant elements [19–21] that impair its function [22,23]. As albumin, via its thiol groups, is the main extracellular antioxidant molecule [24] and ALA-D is a thioldependent enzyme, they can represent better the circulating oxidative status. Vitamin C, in increased levels, probably plays
a protective role against the oxidation of -SH groups in these proteins. Thus, in elderly, higher levels of this nutrient could be necessary for an efficient protection against blood proteins-thiol oxidation verified in the non-institutionalized group due by water-soluble vitamin C characteristic. However, despite the inhibition of thiol groups in albumin, it could be decreased in institutionalized women also due to malnourishment from undetected chronic disease, as previously reported [25]. GSH in its reduced form is accepted as the most important and representative intracellular antioxidant. Reduced GSH is usually located in the red blood cells while its concentration is low in extracellular fluids [26]. On the other hand, MDA is a major product of the free radical attack on polyunsaturated fatty acids and it is widely used as a biomarker of lipid peroxidation [27] indicating the extent of cell membrane injury [28]. In addition, PCO is the most used biomarker for protein oxidative damage, and it reflects the cellular damage induced by multiple forms of ROS [29,30].
Fig. 2. Pearson correlations between vitamin C levels vs ALA-D activity in elder women (n = 67).
Fig. 4. Pearson correlation between ALA-D activity vs age in elder women (n = 67).
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Considering these oxidative stress biomarkers, the differences in the erythrocyte GSH concentration and in serum PCO content were not significant between the groups. However, plasmatic MDA levels of the non-institutionalized group showed a significant increase in relation to the institutionalized ones. However, the MDA levels appear not to have relation with vitamin C levels, maybe because this vitamin is a water-soluble antioxidant. Moreover, other aspects that were not approached in this work also would be contributing to the lower levels of MDA in this group. On the other hand, ALA-D activity was significantly decreased in the institutionalized women. It is known that ALA-D is an enzyme of the heme biosynthesis pathway, essential for all aerobic organisms. It is a zinc metalloenzyme that requires reduced thiol groups for its activity [31]. ALA-D inhibition impairs heme biosynthesis and leads to the accumulation of its intermediates. One of them, δ-aminolevulinic acid (ALA), has been shown to induce pro-oxidant events [32,33]. An interesting and important finding of the present report was the fact that, at the first time in healthy elderly people, ALA-D activity was evaluated such as possible oxidative stress biomarkers. The results showed two interesting aspects: a positive correlation with an important plasma antioxidant, the vitamin C (Fig. 1), and a negative correlation with age and PCO (Fig. 2). The institutionalized and non-institutionalized groups did not present a significant age difference. However, a negative correlation between ALA-D and age was observed (Fig. 3). In opposition, GSH did not present a correlation with age (date not shown). Although it is known that GSH decreases with aging [34,35], this was not found in our study, probably due to the small age variation between both groups. Therefore, these results could suggest that ALA-D may be used to evaluate the relationship between -SH oxidation and aging, but other studies are necessary. Through the correlation with PCO, it was shown that this enzyme could be utilized as an additional protein oxidation marker. In conclusion, the exogenous antioxidant vitamin C presented levels within the normal range for adults. Institutionalized women showed vitamin C, albumin, MDA and ALA-D decrease compared to the non-institutionalized group. Considering the results, we suggest that vitamin C plays a possible protective role, probably avoiding thiolic group oxidation in plasma proteins. Moreover, ALA-D activity was presented as an oxidative stress biomarker in elderly people.
[3]
[4] [5] [6] [7]
[8] [9]
[10] [11]
[12]
[13] [14] [15] [16] [17] [18]
[19]
[20]
[21]
[22]
Acknowledgments [23]
Work supported by CNPq (grant 402243/2005-6 to S.C. Garcia) and DAAD (grant to S.C. Garcia). M. Charão and A. Moro are recipients of the PIBIC Fellowships, respectively. S.C. Garcia is the recipient of CNPq Research Fellowship.
[24]
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