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Effects of vitamin C supplementation on antioxidants and lipid peroxidation markers in elderly subjects with type 2 diabetes
Archives of Gerontology and Geriatrics 48 (2009) 67–72
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Effects of vitamin C supplementation on antioxidants and lipid peroxidation markers in elderly subjects with type 2 diabetes Daniel M. Tessier *, Abdelouahed Khalil, Lise Trottier, Tamas Fu¨lo¨p Biogerontology Laboratory, Research Center on Aging, Sherbrooke Geriatric University Institute, 375 Rue Argyll, Sherbrooke, QC J1J 3H5, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2007 Received in revised form 21 October 2007 Accepted 26 October 2007 Available online 20 February 2008
The objective of our study was to evaluate the effects of the administration of two dosages of vitamin C (Vit-C) (0.5 and 1 g/day, vs. placebo) in elderly patients with type 2 diabetes mellitus on the intracellular levels of Vit-C and glutathione, and on the lipid peroxidation markers and vitamin E (Vit-E) content of low-density lipoprotein (LDL) and on LDL susceptibility to gamma radiolysis-induced peroxidation. Thirty-six patients were randomized into three groups. In patients on 0.5 g Vit-C/day versus the placebo group, a significant increase in cellular reduced glutathione level was observed (0.60 0.26 vs. 0.33 0.27). In patients on 1 g Vit-C/day versus placebo, a significant increase was also observed in cellular reduced glutathione (0.93 0.70 vs. 0.33 0.27), in Vit-C (5.66 2.00 vs. 2.72 1.88) and in vitamin E content of LDL (1.98 0.38 vs. 1.48 0.40). No change was observed in either group in basal levels of lipid peroxidation markers and in the susceptibility of LDL to peroxidation provoked by gamma-radiolysis. In conclusion, Vit-C has a dose-dependent effect on the cellular contents of antioxidants and on vitamin E content of LDL in elderly patients with type 2 DM. These changes are not sufficient to decrease the LDL susceptibility to peroxidation. ß 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Type 2 diabetes Reactive oxygen species Elderly Vitamin C Vitamin E Glutathione
1. Introduction Many human diseases are associated to an increased oxidative stress resulting either from the altered production of free radicals or from the altered antioxidant content or activity. Studies have observed that middle-aged and elderly patients with type 2 DM (T2 DM) have lower circulating levels of vitamin C (Vit-C) and higher levels of oxidative stress markers compared to a control population (Will et al., 1999; Sargeant et al., 2000). Supplementation studies with variable doses of Vit-C in middle-aged (Sinclair et al., 1991; Upritchard et al., 2000) and elderly (Paolisso et al., 1995) patients with T2 DM showed diverging results on plasma concentration of antioxidants. Elderly patients with T2 DM are at high risk to develop atherosclerosis (Kuusisto et al., 1990). One explanation for this phenomenon is an increased susceptibility of low-density lipoproteins (LDL) of these patients to oxidation. LDL from diabetic patients are smaller, have a lower content of vitamin E (Vit-E) and are more susceptible to oxidative modification compared to LDL from non diabetic controls (Yoshida et al., 1997). Biological factors have been demonstrated to be involved in the protection of LDL against oxidation in T2 DM. A study in middle-
aged patients with T2 DM has documented that the administration of Vit-E 800 IU/day for 4 weeks decreased the LDL susceptibility to in vitro oxidation (Upritchard et al., 2000). Oxidation of LDL from normal subjects was prevented and lipid peroxidation was terminated by the addition of 40–60 mM of Vit-C to the LDL preparation (Jialal et al., 1990; Retsky and Frei, 1995). It has been proposed that Vit-C recycles Vit-E by a non-enzymatic reaction. Additional interactions have been also reported between Vit-C and other antioxidants. Vit-C is associated with the recycling of an important cellular antioxidant, the glutathione and functions with it as a redox couple (Winkler et al., 1994). Glutathione is also involved in the recycling of Vit-E by an enzymatic mechanism (McCay, 1985; Chan, 1993). Thus, antioxidants form an interacting system where one antioxidant may modulate the activity of many others. The goal of this study was to investigate the dose-dependent effects of Vit-C administration on cellular content in antioxidants, levels of lipid peroxidation markers and susceptibility of LDL to oxidation in an elderly population with T2 DM. 2. Materials and methods 2.1. Subjects
* Corresponding author. Tel.: +1 819 821 1170x3254; fax: +1 819 829 7145. E-mail address: [email protected] (D.M. Tessier). 0167-4943/$ – see front matter ß 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2007.10.005
Candidates for this study were recruited from a list of patients who previously had attended the Diabetes Day Care Center
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activities of the Centre Hospitalier Universitaire de SherbrookeFleurimont site and were required to be older than 65 years old at randomization, ambulatory outpatients with no acute cardiovascular or neurologic event in the prior 6 months, no history of active tobacco smoking and no oral intake of vitamin supplements in the last 4 weeks. Medical treatment for diabetes had to be stable for the last 3 months with an HbA1c 9%. The study protocol was approved by the Ethics Committee of the Research Centre on Aging. All subjects provided written informed consent before participating in this study. 2.2. Procedure After baseline assessment for eligibility, each subject was randomized in a double-blind fashion to either placebo, Vit-C 0.5 or 1.0 g once a day in the morning for a total of 12 weeks. Vit-C tablets were purchased from the Wampole Company (Toronto, Canada). Each subject met a registered dietician at baseline and week 12 visits to assess the dietary intake of Vit-C. Four 24 h recalls were conducted as suggested by Madden et al. (1976) for elderly subjects (Block, 1982). The NutriPro-GI program was used for analysis of nutritional data (Glycaemic Index Testing Inc., Toronto, Canada). The following measurements were done at baseline and at week 12: HbA1c, glycemia, creatinine and a standard lipid profile. All blood work was done fasting in the morning, 24 h after the last oral dose of Vit-C. The glucose level was measured by a glucose oxidase colorimetric method (Vitros; Johnson and Johnson Clinical Diagnosis, Rochester, USA), HbA1c (labile fraction removed) by fast protein liquid chromatography (Pharmacia, Uppsala, Sweden) and standard lipid profile by an enzymatic and colorimetric method (Roche Diagnostic Systems, Mississauga, Canada). The LDL level was calculated using the Friedwald equation. 2.3. Basal oxidative stress Basal oxidative stress was evaluated by measuring conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) as well as Vit-E content of LDL and high-density lipoproteins (HDL) after their isolation by ultracentrifugation. We also measured cellular levels of Vit-C, reduced (GSH) and oxidized (GSSG) glutathione in granulocytes. Acetic acid, sulfuric acid, n-butanol, sodium phosphate, thiobarbituric acid, methanol, hexane, monobasic potassium phosphate, potassium iodide and sodium azide were purchased from Fisher Scientific (Montre´al, QC). 1,1,3,3-tetraethoxypropane, ethylene glycol-bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), benzalkonium chloride, ammonium molybdate, butylated hydroxy toluene (BHT) were obtained from Sigma (St. Louis, MO) and Triton X-100 from ICN Biochemicals (Aurora, OH). Dialysis bags were purchased from Spectrum Medical Industries Inc. (Houston, TX). 2.4. Blood collection and processing Blood was collected after overnight fasting. Forty milliliters of blood were collected in EDTA for lipoprotein isolation and 40 ml were collected in heparin for granulocyte (PMNs) separation. PMNs isolation was carried out by Fycoll Hypaque gradient centrifugation as previously described (Fortin et al., 2007). The PMNs (2 106/ml) were suspended in 200 mM H3PO4 (pH 3) containing 0.1 mM EDTA to minimize autoxidation and then were lysed by three freeze/thaw and spun for 5 min (13,000 rpm) in a microcentrifuge. Their supernatants were conserved at 80 8C until ascorbate and GSH/GSSG analysis.
2.5. Antioxidant measurements Cellular levels of Vit-C were determined by high-performance liquid chromatography (HPLC) with electrochemical detection (CSC hypersil column 12OA/ODS, 5 mM, 25 cm 0.43 cm). The mobile phase was 200 mM H3PO4, pH 3, eluted at a rate of 1.2 ml/ min (Rose and Bode, 1995; Bode and Rose, 1999). Standards were prepared from frozen stock (70 8C). GSH/GSSG were extracted in 200 mM H3PO4 in the supernatant by enzymatic methods using ophthalaldehyde (OPT) as a fluorescent reagent (Hissin and Hilf, 1976). For GSSG analysis, 40 mM of N-ethylmaleimide (NEM) was added prior to extraction to neutralize GSH autoxidation. Endogenous Vit-E in LDL and HDL was assayed as alpha-tocopherol by reverse phase HPLC, with UV (292 nm) and electrochemical detections as already described (De Leenher et al., 1978; Khalil et al., 1998a,b). Alpha-tocopherol was assayed on Sephasil Peptide column (C18 5 mm ST 4.6/250) (Pharmacia Biotech.). The elution phase (1.2 ml/min) was a solvent mixture of methanol, ethanol and isopropanol (88/24/10, v/v/v) containing lithium perchlorate at concentration 20 mM. 2.6. Lipoprotein isolation LDL and HDL were isolated within 2 h of collection from human plasma collected in EDTA (0.4 g/l) by ultracentrifugation (Sattler et al., 1994; Khalil et al., 1998a,b). Density of plasma was increased to 1.2 g/ml by adding KBr and then overlayered with PBS (d = 1.007 g/ml) and spun at 1.0 105 rpm (5.1 105 g) in a beckman TLA 100.4 rotor for 2 h at 15 8C before the distinct LDL and HDL bands were collected by syringe. Isolated LDL and HDL were dialyzed overnight at 4 8C with 102 M sodium phosphate buffer (pH 7) with argon bubbling at a rate of 20 ml/min through the dialysis solution (4 l changed twice) to ensure anaerobic conditions. Concentrations of LDL and HDL solutions were given in terms of total protein concentrations (100 mg/ml). Proteins were measured by commercial assay (Pierce method, Rockford, IL). 2.7. Lipoprotein oxidation Lipoprotein oxidation was induced by oxygen free radicals produced by gamma-radiolysis as previously described (Khalil et al., 1995, 2000). Briefly, lipoproteins were irradiated in oxygenated aqueous solutions containing 102 M sodium phosphate buffer at pH 7. In our experimental system, irradiation of aqueous solutions with ionizing radiation (gamma-rays of 60Co in our case) leads to the formation of two major initial free radicals: hydroxyl radicals (OH), hydrogen atoms (H) and hydrated electrons (eaq) (Fricke and Morse, 1927). In the presence of oxygen, eaq and H are transformed quantitatively into superoxide anions (O2) and perhydroxyl radicals (HO2), respectively. HO2 is the acidic form of O2 (pKa 5.7) (Spinks and Woods, 1990). The radiolytic yields of O2 and OH formation under our conditions were 3.4 107 and 2.8 107 mol/J. Gamma irradiations were carried out using a 60Co Gamma cell 220 (Atomic Energy of Canada Ltd.) as previously described (Khalil et al., 1998a,b). The dose rate was 0.13 Gy/s as determined by Fricke dosimetry (Fricke and Morse, 1927). Doses of radiation used were 20, 40, 80, 120, 160, and 200 Gy. LDL oxidation induced by free radicals produced by gamma-radiolysis was monitored as follows: CD formation, irradiated lipoproteins (LDL and HDL) (100 mg/ml) were continuously monitored at 234 nm (Khalil et al., 1998a,b) to detect the formation of CD as previously described (Khalil et al., 2000). TBARS formation was assayed as described by Yagi (1976), but without precipitation with phosphotungstic acid (Bonnefont-
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Rousselot et al., 1995). The coefficient of variation of this assay is less than 10% (Khalil et al., 2000). TBARS concentrations were calculated as malondialdehyde (MDA) equivalents using the MDA standard curve. MDA was generated by the hydrolysis of 1,1,3,3tetraethoxypropane. 2.8. Data analysis Given the small size of our experimental sample, nonparametric procedures were used. For between-group comparisons, continuous variables were analyzed using the Kruskall–Wallis test for independant samples. Fisher’s exact test was used to compare the groups on categorical variables (gender). Within-group comparisons were made with the Wilcoxon signed rank test. Data on LDL and HDL irradiation curves were analyzed using an ANOVA method with repeated measurement. All data are presented as the mean S.D. The alpha level of statistical tests was set at 0.05 unless specified otherwise.
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Table 1 Population description Parameter/groups
Placebo
0.5 g Vit-C
1.0 g Vit-C
N Age (years) Female/male Duration diabetes (years) Body mass index (kg/m2) Fasting glycemia (mmol/l) HbA1c (%) Total cholesterol (mmol/l) LDL (mmol/l) HDL (mmol/l) Triglycerides (mmol/l) Creatinine (mmol/l) Dietary Vit-C (mg/day)
12 71 4 10/2 7.7 8.9 28.9 1.9 8.4 1.6 7.3 0.8 5.22 0.92 2.93 0.84 1.08 0.34 2.67 0.81 94 15 86 46
12 72 5 9/3 10.5 11.8 29.8 3.7 8.2 2.2 7.4 0.9 4.64 0.93 2.72 0.82 1.1 0.34 2.12 1.26 90 36 89 60
12 72 4 9/3 10.2 9.4 29.5 5.3 8.3 2.9 7.4 0.8 4.63 0.76 2.51 0.80 1.02 0.3 2.43 0.83 87 18 78 41
Daily dose Glibenclamide (mg/day) Metformin (g/day) Insulin (units/day)
13.6 6.9 (7)a 1.5 0.4 (5) 84 (1)
8.8 5.4 (6) 1.2 0.6 (9) 30 (1)
11.8 7.4 (11) 1.5 0.6 (9) 57 38 (2)
a Number between parenthesis = number of patients/group under this medication.
3. Results 3.4. Llipid peroxidation markers and susceptibility of LDL and HDL to oxidation induced by gamma-radiolysis
3.1. Baseline data Table 1 shows the demographic characteristics, the biochemical parameters and the daily dietary intake of Vit-C of the 36 subjects who participated in our study. Considering the parameters reported in Table 1, no difference was observed between groups at baseline and within groups at week 12. 3.2. Vit-E content of LDL and HDL Vit-E content of LDL and HDL was similar between groups at baseline. Vit-E content of LDL was significantly increased in subjects treated with 1 g of Vit-C at week 12 (p < 0.05) (Table 2). No significant change in Vit-E content of LDL was observed in subjects under placebo or Vit-C 0.5 g. Vit-E content of HDL was similar between groups at baseline. Vit-C administration at any doses had no effect on the Vit-E content of HDL. 3.3. Cellular level of Vit-C and glutathione At week 12, cellular levels of Vit-C were significantly higher in the 0.5 and 1.0 g groups compared to placebo (p < 0.05). However, the within-group comparisons demonstrated a significant increase of Vit-C only in the 1.0 g group between baseline and week 12 (p < 0.01). For cellular levels of GSH, a significant increase was observed in the 0.5 and the 1.0 groups compared to baseline (p < 0.05) and compared to placebo (p < 0.01) (Table 3). No change was observed for cellular levels of GSSG in the three groups before and after Vit-C supplementation.
Despite an increased content in Vit-E in LDL particles of patients taking 1.0 g of Vit-C (Table 2), no significant change in basal levels of markers of lipid peroxidation (TBARS and CD) in LDL and HDL was observed between baseline and week 12 in the three experimental groups (Table 4). Curves of TBARS and CD generated by gamma-radiolysis of LDL and HDL were analyzed in regard to group and dose/group effects at baseline and at week 12 with an ANOVA method (Table 5). At week 12, no significant difference was detected for these markers in the two Vit-C groups compared to placebo. 4. Discussion Previous studies have suggested that T2 DM is a disease in which reactive oxygen species (ROS) are involved in the pathogenesis of complications (Brownlee, 2001). An epidemiologic study has observed that plasma levels of the antioxidant Vit-C were inversely correlated with HbA1c in diabetic patients (Will et al., 1999). However, in middle-aged subjects with T2 DM, this abnormality is only partially corrected with an oral supplementation of 1 g daily Vit-C (Sinclair et al., 1991). We recently documented that in elderly patients with type 2 DM, hyperglycemia following a meal induces a depletion of Vit-C and an accumulation of GSSG at cellular level (Tessier et al., 1999a). It has been observed that LDL from subjects with T2 DM are more susceptible to oxidation (Yoshida et al., 1997). To normal subjects and to patients with T2 DM, the supplementation of Vit-E
Table 2 Vit-E content of LDL and HDL Group
Placebo
0.5 g Vit-C
1.0 g Vit-C
LDL (mM) Baseline Week 12
1.27 0.51 (7.0 2.8)a 1.48 0.40 (8.2 2.2)
1.31 0.54 (7.2 3.0) 1.57 0.47 (8.7 2.6)
1.23 0.41 (6.8 2.3) 1.98 0.38b,c (10.9 2.1)
HDL (mM) Baseline Week 12
0.55 0.19 (3.1 1.1) 0.60 0.33 (3.3 1.8)
0.52 0.18 (2.9 1.0) 0.60 0.28 (3.3 1.5)
0.46 0.17 (2.6 1.0) 0.63 0.20 (3.5 1.1)
a b c
In parenthesis: number of Vit-E molecules/LDL or HDL particle. p < 0.05 vs. baseline. p < 0.05 vs. placebo group.
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70 Table 3 Cellular levels of Vit-E and glutathione Groups
0.5 g Vit-C
1.0 g Vit-C
Vit-C (nmol/mg prot) Baseline 2.42 2.01 Week 12 2.72 1.88
3.36 1.99 5.39 1.90a
1.45 1.20 5.66 2.00a,b
GSH (nmol/mg prot) Baseline 0.50 0.70 Week 12 0.33 0. 27
0.36 0.23 0.60 0.26c,d
0.37 0.40 0.93 0.70c,d
GSSG (nmol/mg prot) Baseline 0.18 0.27 Week 12 0.33 0.43
0.34 0.28 0.36 0.14
0.26 0.28 0.29 0.27
a b c d
Placebo
p < 0.01 vs. placebo. p < 0.01 vs. baseline. p < 0.01 vs. placebo. p < 0.05 vs. baseline.
protected their LDL against in vitro peroxidation (Jialal et al., 1995; Upritchard et al., 2000). It has been proposed that recycling of Vit-E is dependent on Vit-C (Chan, 1993). In vitro studies observed that the addition of 40–60 mM of Vit-C can prevent initiation and terminate lipid peroxidation in LDL by recycling Vit-E (Retsky and Frei, 1995; Tribble et al., 1995). Vit-E is distributed in the core and in the envelope of the LDL (Cazzola et al., 1999). It has been proposed that the hydrophilic properties of Vit-C facilitate their localization at the interface of the lipid bilayers in membranes, Table 4 TBARS and CD contents of LDL and HDL Group
Placebo
0.5 g Vit-C
1.0 g
LDL Baseline Week 12
0.13 0.19 0.11 0.14
0.13 0.17 0.27 0.20
0.27 0.19 0.17 0.19
HDL Baseline Week 12
0.36 0.75 0.18 0.25
0.13 0.16 0.27 0.29
0.11 0.13 0.11 0.16
0.27 0.12 0.24 0.09
0.23 0.05 0.23 0.11
0.20 0.06 0.27 0.11
0.15 0.11 0.16 0.08
0.14 0.04 0.13 0.05
0.12 0.06 0.21 0.11
TBARS (mmol/l)
CD (OD at 234 nm) LDL Baseline Week 12 HDL Baseline Week 12
Table 5 ANOVA table TBARS and CD contents of LDL and HDL under gamma-radiolysis Effect
Group
Dose group
TBARS (mmol/l) LDL Baseline Week 12
0.39 0.37
0.44 0.70
HDL Baseline Week 12
0.61 0.50
0.50 0.69
0.90 0.03
0.71 0.16
0.46 0.10
0.67 0.41
CD (OD at 234 nm) LDL Baseline Week 12 HDL Baseline Week 12
thereby suggesting two advantages: (i) effective inhibition of attack by free radicals in the aqueous phase, and (ii) effective repair of lipophilic antioxidants (Laranjinha and Cadenas, 1999). Our observation that the in vivo administration of 1 g of Vit-C daily increases Vit-E content of LDL adds one more argument that Vit-C seems to positively modulate Vit-E levels by being moderately protective for Vit-E. In an in vitro model comparing the susceptibility to oxidation of dense versus buoyant LDL, addition of Vit-C resulted in a significant prolongation of the lag time for the 50% depletion of Vit-E in the dense LDL compared to the buoyant ones (Tribble et al., 1995). This result is especially pertinent in that patients with T2 DM have smaller LDL which are more susceptible to in vitro oxidation (Yoshida et al., 1997). Despite the in vitro effect of Vit-C on susceptibility of LDL to oxidation, a clinical study in middleaged patients with type 2 DM failed to show any effect of an oral daily dose of 0.5 g of Vit-C on the susceptibility of LDL to oxidation (Upritchard et al., 2000). Our results seem to confirm the data obtained in this latter study. However, regarding our results concerning the lack of change of LDL susceptibility to oxidation by gamma-radiolysis, we should take into account the magnitude of the effect of oral supplementation in Vit-C in regard to the Vit-E content of LDL. In a dose–response study with Vit-E, the Vit-E content of LDL increased by as much as 175% with the 1200 IU/day dosage (Jialal et al., 1995). In our study, the size of the effect was +61% for Vit-E content in LDL of patients taking 1 g daily of Vit-C. There are no data whether higher doses of Vit-C administration would increase more the LDL content of this vitamin. This needs further studies. Moreover, it should be noted that in the process of LDL isolation, ultracentrifugation removes the plasma containing the antioxidants such as Vit-C and glutathione that are usually present in the biological environment of the lipid particles. HDL is involved in the protection of LDL against lipid peroxidation via the enzymes paraoxonase and platelet acetylating factor acetylhydrolase (Watson et al., 1995; Hedrick et al., 2000; Berrougui et al., 2007), and glycation diminishes paraoxonase activity (Mackness et al., 1998). We observed that products of lipid peroxidation can be measured in HDL of patients with type 2 DM (Tessier et al., 1999b). An epidemilogical study observed that each 1 mg/dl increase in serum Vit-C was independently associated with an increase of 2–3 mg/dl in HDL-C level (Simon and Hudes, 1998), but no clinical study yet, including our present one, documented that oral supplementation of Vit-C could diminish basal levels of lipid peroxidation products present in HDL and susceptibility of this particle to peroxidation. GSH, a tripeptide containing a sulfhydryl group, plays a key role in detoxification of ROS. Observational studies demonstrated a correlation of Vit-C and glutathione in normal elderly subjects (Lenton et al., 2000). It has been suggested in the literature that VitC and glutathione function as a redox couple (Winkler et al., 1994). When Vit-C is oxidized to dehydroascorbate (DHAA), a glutathione-dependent DHAA-reductase activity may be important in the physiologic recycling of Vit-C (Vethanayagan et al., 1999). Under experimental oxidative stress with hydrogen peroxide, VitC may preferentially react with the ROS and by doing so, preserves the reserve in glutathione (Sturgeon et al., 1998). One animal study demonstrated that increasing dietary content in Vit-E resulted in a gradual increase in the GSH/GSSG ratio and supplementation with 100 IU Vit-E/day in children with type 1 diabetes increased erythrocyte level of glutathione (Jain et al., 2000). These observations suggest a protecting role of Vit-E on GSH and may be proposed as elements to explain the increase in the cellular levels of GSH that we observed in our present study. In addition, GSH regenerates Vit-E in vitro with the aid of protein catalysis (McCay, 1985; Chan, 1993). Our results demonstrating that Vit-C
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supplementation can increase the cellular Vit-C and GSH levels suggest that it is possible to modulate the neutrophils and possibly, other cell antioxidant content in a context of oxidative stress such as T2 DM. This could confer a better functioning and a better innate immune defense in these cells. Altogether these data show that it exists an interacting network between various antioxidants modulating the content and activity of each other. Recent studies have suggested additional interactions between Vit-C and T2 DM. In an observational study, an inverse relationship between Vit-C levels and markers of DNA damages (Choi et al., 2005) has been demonstrated. In human mononuclear cells involved in the atherogenic process, Vit-C down regulates the genes associated with proliferation (Kaul and Baba, 2005). In patients with T2 DM and coronary artery disease, Vit-C supplementation for 4 weeks increases forearm blood flow (Tousoulis et al., 2003). The mechanism of this increment in blood flow seems to be independent of nitric oxide (Ajay and Mustafa, 2006). In summary, oral supplementation with Vit-C in elderly patients with T2 DM, increases cellular reserve in antioxidants namely Vit-C and glutathione, and the Vit-E content of LDL in a dose-dependent manner. The lack of effect of Vit-C on lipid peroxidation markers and susceptibility of lipid particles to oxidation is probably due to the relatively small size of the effect of Vit-C on Vit-E. Our study confirms the in vivo interactions between antoxidants but the clinical benefits of these changes remains unclear. We still lack clinical data for clear-cut recommendation for the use of antioxidants in various clinical settings. Nevertheless, studies such as ours contribute for the better understanding of the in vivo effects of Vit-C in special clinical settings such as T2 DM. More controlled clinical trials are needed to fully elucidate the effects of antioxidants including Vit-C on cellular and molecular markers of oxidative stress and on disease pathogenesis and progression. References Ajay, M., Mustafa, M.R., 2006. Effects of ascorbic acid on impaired vascular reactivity in aortas isolated from age-matched hypertensive diabetic rats. Vascul. Pharmacol. 45, 127–144. Berrougui, H., Isabelle, M., Cloutier, M., Grenier, G., Khalil, A., 2007. Age-related impairment of HDL-mediated cholesterol efflux. J. Lipid Res. 48, 328–336. Block, G., 1982. A review of validations of dietary assessment methods. Am. J. Epidemiol. 115, 492–505. Bode, A.M., Rose, R.C., 1999. Analysis of water-soluble antioxidants by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 299, 77–83. Bonnefont-Rousselot, D., Motta, C., Khalil, A., Sola, R., La Ville, A.E., Delattre, J., Gardes-Albert, M., 1995. Physicochemical changes in human high-density lipoproteins (HDL) oxidized by gamma radiolysis-generated oxyradicals. Effect on their cholesterol effluxing capacity. Biochim. Biosphys. Acta 1255, 23–30. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820. Cazzola, R., Cervato, G., Cestaro, B., 1999. Variability in alpha-tocopherol antioxidants activity in the core and surface layers of low- and high-density lipoproteins. J. Nutr. Sci. Vitaminol. 45, 39–48. Chan, A.C., 1993. Partners in defense, vitamin E and vitamin C. Can. J. Physiol. Pharmacol. 71, 725–731. Choi, S.W., Benzie, I.F., Lam, C.S., Chat, S.W., Lam, J., Yiu, C.H., Kwan, J.J., Tang, Y.H., Yeung, G.S., Yeung, V.T., Woo, G.C., Hannigan, B.M., Strain, J.J., 2005. Interrelationships between DNA damage, ascorbic acid and glycaemic control in type 2 diabetes mellitus. Diabet. Med. 22, 1347–1353. De Leenher, A.P., De Bevere, V.O., Cruyl, A.A., Claeys, A.E., 1978. Determination of serum alpha-tocopherol (vitamin E) by high-performance liquid chromatography. Clin. Chem. 24, 585–590. Fortin, C.F., Lesur, O., Fulop, T., 2007. Effects of aging on triggering receptor expressed on myeloid cells (TREM)-1-induced PMN functions. FEBS Lett. 581, 1173–1178. Fricke, H., Morse, S., 1927. The chemical action of Ro¨ntgen rays on dilute ferrosulfate solutions as a measure of dose. Am. J. Roentgen Radiat. Ther. 18, 430–432. Hedrick, C.C., Thorpe, S.R., Fu, M.-X., Harper, C.M., Yoo, J., Kim, S.-M., Wong, H., Peters, A.L., 2000. Glycation impairs high-density lipoprotein function. Diabetologia 43, 312–320. Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214–226.
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Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low density lipoproteins against oxidation. Life 48, 57–65. Lenton, K.J., Therriault, H., Cantin, A.M., Fu¨lo¨p, T., Payette, H., Wagner, J.R., 2000. Direct correlation of glutathione and ascorbate and their dependence on age and season in human lymphocyte. Am. J. Clin. Nutr. 71, 1120–1194. Mackness, B., Mackness, M.I., Arrol, S., Turkie, W., Julier, K., Abuasha, B., Miller, J.E., Boulton, A.J., Durrington, P.N., 1998. Serum paraoxonase (PON1) 55 and 192 polymorphism and paraoxonase activity and concentration in non-insulindependent diabetes mellitus. Atherosclerosis 139, 341–349. Madden, J.P., Goodman, S.J., Guthrie, H.A., 1976. Validity of the 24-hour recall. Analysis of data obtained from elderly subjects. J. Am. Diet. Assoc. 68, 143–147. McCay, P.B., 1985. Vitamin E: interaction with free radicals and ascorbate. Annu. Rev. Nutr. 5, 323–340. Paolisso, G., Balbi, V., Volpe, C., Varricchio, G., Gambardella, A., Saccomanno, F., Ammendola, S., Varricchio, M., D’Onofrio, F., 1995. Metabolic benefits deriving from chronic vitamin C supplementation in aged non-insulin dependent diabetics. J. Am. Coll. Nutr. 14, 387–392. Retsky, K.L., Frei, B., 1995. Vitamin C prevents metal ion-dependent and propagation of lipid peroxidation in human low-density lipoprotein. Biochim. Biophys. Acta 1257, 279–287. Rose, R., Bode, A.M., 1995. Analysis of water soluble antioxidants by high pressure liquid chromatography. Biochem. J. 306, 101–105. Sargeant, L.A., Wareham, N.J., Bingham, S., Day, N.E., Luben, R.N., Oakes, S., Welch, A., Khaw, K., 2000. Vitamin C and hyperglycemia in the European Prospective Investigation into Cancer-Norfolk (EPIC-Norfolk) study. Diabetes Care 23, 726– 732. Sattler, W., Mohr, D., Stocker, R., 1994. Rapid isolation of lipoproteins and assessment of their peroxidation status by high-performance liquid chromatography postcolumn chemiluminescence. Methods Enzymol. 233, 469–489. Simon, J.A., Hudes, E.S., 1998. Relation of serum ascorbic acid to serum lipids and lipoproteins in US adults. J. Am. Coll. Nutr. 17, 250–255. Sinclair, A.J., Girling, A.J., Gray, L., Le Guen, C., Lunec, J., Barnett, A.H., 1991. Disturbed handling of ascorbic acid in diabetic patients with and without microangiopathy during high dose ascorbate supplementation. Diabetologia 34, 171– 175. Spinks, J.W.T., Woods, R.J., 1990. An Introduction to Radiation Chemistry, 3rd ed. Wiley, New York, pp. 243–313. Sturgeon, B.E., Sipe H.J.Jr., Barr, D.P., Corbett, J.T., Martinez, J.G., Mason, R.P., 1998. The fate of oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis. J. Biol. Chem. 273, 30116– 30121. Tessier, D., Khalil, A., Fu¨lo¨p, T., 1999a. Effects of an oral glucose challenge on free radicals/antioxidants balance in an older population with type II diabetes. J. Gerontol. A, Biol. Sci. Med. Sci. 11, M541–M545. Tessier, D., Maheux, P., Khalil, A., Fu¨lo¨p, T., 1999b. Effects of gliclazide versus metformin on the clinical profile and lipid peroxidation markers in type 2 diabetes. Metabolism 48, 897–903. Tousoulis, D., Antoniades, C., Tountas, C., Bosinakou, E., Kotsopoulou, M., Toutouzas, P., Stefanadis, C., 2003. Vitamin C affects thrombolysis/fibrinolysis systems and reactive hyperemia in patients with type 2 diabetes and coronary artery disease. Diabetes Care 26, 2749–2753. Tribble, D.L., Thiel, P.M., Van den Berg, J.M., Krauss, R.M., 1995. Differing alphatocopherol oxidative lability and sparing effects in buoyant and dense LDL. Arterioscler. Thromb. Vasc. Biol. 15, 2025–2031. Upritchard, J.E., Sutherland, W.H.E., Mann, J.I., 2000. 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Effects of vitamin C supplementation on antioxidants and lipid peroxidation markers in elderly subjects with type 2 diabetes Daniel M. Tessier *, Abdelouahed Khalil, Lise Trottier, Tamas Fu¨lo¨p Biogerontology Laboratory, Research Center on Aging, Sherbrooke Geriatric University Institute, 375 Rue Argyll, Sherbrooke, QC J1J 3H5, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2007 Received in revised form 21 October 2007 Accepted 26 October 2007 Available online 20 February 2008
The objective of our study was to evaluate the effects of the administration of two dosages of vitamin C (Vit-C) (0.5 and 1 g/day, vs. placebo) in elderly patients with type 2 diabetes mellitus on the intracellular levels of Vit-C and glutathione, and on the lipid peroxidation markers and vitamin E (Vit-E) content of low-density lipoprotein (LDL) and on LDL susceptibility to gamma radiolysis-induced peroxidation. Thirty-six patients were randomized into three groups. In patients on 0.5 g Vit-C/day versus the placebo group, a significant increase in cellular reduced glutathione level was observed (0.60 0.26 vs. 0.33 0.27). In patients on 1 g Vit-C/day versus placebo, a significant increase was also observed in cellular reduced glutathione (0.93 0.70 vs. 0.33 0.27), in Vit-C (5.66 2.00 vs. 2.72 1.88) and in vitamin E content of LDL (1.98 0.38 vs. 1.48 0.40). No change was observed in either group in basal levels of lipid peroxidation markers and in the susceptibility of LDL to peroxidation provoked by gamma-radiolysis. In conclusion, Vit-C has a dose-dependent effect on the cellular contents of antioxidants and on vitamin E content of LDL in elderly patients with type 2 DM. These changes are not sufficient to decrease the LDL susceptibility to peroxidation. ß 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Type 2 diabetes Reactive oxygen species Elderly Vitamin C Vitamin E Glutathione
1. Introduction Many human diseases are associated to an increased oxidative stress resulting either from the altered production of free radicals or from the altered antioxidant content or activity. Studies have observed that middle-aged and elderly patients with type 2 DM (T2 DM) have lower circulating levels of vitamin C (Vit-C) and higher levels of oxidative stress markers compared to a control population (Will et al., 1999; Sargeant et al., 2000). Supplementation studies with variable doses of Vit-C in middle-aged (Sinclair et al., 1991; Upritchard et al., 2000) and elderly (Paolisso et al., 1995) patients with T2 DM showed diverging results on plasma concentration of antioxidants. Elderly patients with T2 DM are at high risk to develop atherosclerosis (Kuusisto et al., 1990). One explanation for this phenomenon is an increased susceptibility of low-density lipoproteins (LDL) of these patients to oxidation. LDL from diabetic patients are smaller, have a lower content of vitamin E (Vit-E) and are more susceptible to oxidative modification compared to LDL from non diabetic controls (Yoshida et al., 1997). Biological factors have been demonstrated to be involved in the protection of LDL against oxidation in T2 DM. A study in middle-
aged patients with T2 DM has documented that the administration of Vit-E 800 IU/day for 4 weeks decreased the LDL susceptibility to in vitro oxidation (Upritchard et al., 2000). Oxidation of LDL from normal subjects was prevented and lipid peroxidation was terminated by the addition of 40–60 mM of Vit-C to the LDL preparation (Jialal et al., 1990; Retsky and Frei, 1995). It has been proposed that Vit-C recycles Vit-E by a non-enzymatic reaction. Additional interactions have been also reported between Vit-C and other antioxidants. Vit-C is associated with the recycling of an important cellular antioxidant, the glutathione and functions with it as a redox couple (Winkler et al., 1994). Glutathione is also involved in the recycling of Vit-E by an enzymatic mechanism (McCay, 1985; Chan, 1993). Thus, antioxidants form an interacting system where one antioxidant may modulate the activity of many others. The goal of this study was to investigate the dose-dependent effects of Vit-C administration on cellular content in antioxidants, levels of lipid peroxidation markers and susceptibility of LDL to oxidation in an elderly population with T2 DM. 2. Materials and methods 2.1. Subjects
* Corresponding author. Tel.: +1 819 821 1170x3254; fax: +1 819 829 7145. E-mail address: [email protected] (D.M. Tessier). 0167-4943/$ – see front matter ß 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2007.10.005
Candidates for this study were recruited from a list of patients who previously had attended the Diabetes Day Care Center
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activities of the Centre Hospitalier Universitaire de SherbrookeFleurimont site and were required to be older than 65 years old at randomization, ambulatory outpatients with no acute cardiovascular or neurologic event in the prior 6 months, no history of active tobacco smoking and no oral intake of vitamin supplements in the last 4 weeks. Medical treatment for diabetes had to be stable for the last 3 months with an HbA1c 9%. The study protocol was approved by the Ethics Committee of the Research Centre on Aging. All subjects provided written informed consent before participating in this study. 2.2. Procedure After baseline assessment for eligibility, each subject was randomized in a double-blind fashion to either placebo, Vit-C 0.5 or 1.0 g once a day in the morning for a total of 12 weeks. Vit-C tablets were purchased from the Wampole Company (Toronto, Canada). Each subject met a registered dietician at baseline and week 12 visits to assess the dietary intake of Vit-C. Four 24 h recalls were conducted as suggested by Madden et al. (1976) for elderly subjects (Block, 1982). The NutriPro-GI program was used for analysis of nutritional data (Glycaemic Index Testing Inc., Toronto, Canada). The following measurements were done at baseline and at week 12: HbA1c, glycemia, creatinine and a standard lipid profile. All blood work was done fasting in the morning, 24 h after the last oral dose of Vit-C. The glucose level was measured by a glucose oxidase colorimetric method (Vitros; Johnson and Johnson Clinical Diagnosis, Rochester, USA), HbA1c (labile fraction removed) by fast protein liquid chromatography (Pharmacia, Uppsala, Sweden) and standard lipid profile by an enzymatic and colorimetric method (Roche Diagnostic Systems, Mississauga, Canada). The LDL level was calculated using the Friedwald equation. 2.3. Basal oxidative stress Basal oxidative stress was evaluated by measuring conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) as well as Vit-E content of LDL and high-density lipoproteins (HDL) after their isolation by ultracentrifugation. We also measured cellular levels of Vit-C, reduced (GSH) and oxidized (GSSG) glutathione in granulocytes. Acetic acid, sulfuric acid, n-butanol, sodium phosphate, thiobarbituric acid, methanol, hexane, monobasic potassium phosphate, potassium iodide and sodium azide were purchased from Fisher Scientific (Montre´al, QC). 1,1,3,3-tetraethoxypropane, ethylene glycol-bis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), benzalkonium chloride, ammonium molybdate, butylated hydroxy toluene (BHT) were obtained from Sigma (St. Louis, MO) and Triton X-100 from ICN Biochemicals (Aurora, OH). Dialysis bags were purchased from Spectrum Medical Industries Inc. (Houston, TX). 2.4. Blood collection and processing Blood was collected after overnight fasting. Forty milliliters of blood were collected in EDTA for lipoprotein isolation and 40 ml were collected in heparin for granulocyte (PMNs) separation. PMNs isolation was carried out by Fycoll Hypaque gradient centrifugation as previously described (Fortin et al., 2007). The PMNs (2 106/ml) were suspended in 200 mM H3PO4 (pH 3) containing 0.1 mM EDTA to minimize autoxidation and then were lysed by three freeze/thaw and spun for 5 min (13,000 rpm) in a microcentrifuge. Their supernatants were conserved at 80 8C until ascorbate and GSH/GSSG analysis.
2.5. Antioxidant measurements Cellular levels of Vit-C were determined by high-performance liquid chromatography (HPLC) with electrochemical detection (CSC hypersil column 12OA/ODS, 5 mM, 25 cm 0.43 cm). The mobile phase was 200 mM H3PO4, pH 3, eluted at a rate of 1.2 ml/ min (Rose and Bode, 1995; Bode and Rose, 1999). Standards were prepared from frozen stock (70 8C). GSH/GSSG were extracted in 200 mM H3PO4 in the supernatant by enzymatic methods using ophthalaldehyde (OPT) as a fluorescent reagent (Hissin and Hilf, 1976). For GSSG analysis, 40 mM of N-ethylmaleimide (NEM) was added prior to extraction to neutralize GSH autoxidation. Endogenous Vit-E in LDL and HDL was assayed as alpha-tocopherol by reverse phase HPLC, with UV (292 nm) and electrochemical detections as already described (De Leenher et al., 1978; Khalil et al., 1998a,b). Alpha-tocopherol was assayed on Sephasil Peptide column (C18 5 mm ST 4.6/250) (Pharmacia Biotech.). The elution phase (1.2 ml/min) was a solvent mixture of methanol, ethanol and isopropanol (88/24/10, v/v/v) containing lithium perchlorate at concentration 20 mM. 2.6. Lipoprotein isolation LDL and HDL were isolated within 2 h of collection from human plasma collected in EDTA (0.4 g/l) by ultracentrifugation (Sattler et al., 1994; Khalil et al., 1998a,b). Density of plasma was increased to 1.2 g/ml by adding KBr and then overlayered with PBS (d = 1.007 g/ml) and spun at 1.0 105 rpm (5.1 105 g) in a beckman TLA 100.4 rotor for 2 h at 15 8C before the distinct LDL and HDL bands were collected by syringe. Isolated LDL and HDL were dialyzed overnight at 4 8C with 102 M sodium phosphate buffer (pH 7) with argon bubbling at a rate of 20 ml/min through the dialysis solution (4 l changed twice) to ensure anaerobic conditions. Concentrations of LDL and HDL solutions were given in terms of total protein concentrations (100 mg/ml). Proteins were measured by commercial assay (Pierce method, Rockford, IL). 2.7. Lipoprotein oxidation Lipoprotein oxidation was induced by oxygen free radicals produced by gamma-radiolysis as previously described (Khalil et al., 1995, 2000). Briefly, lipoproteins were irradiated in oxygenated aqueous solutions containing 102 M sodium phosphate buffer at pH 7. In our experimental system, irradiation of aqueous solutions with ionizing radiation (gamma-rays of 60Co in our case) leads to the formation of two major initial free radicals: hydroxyl radicals (OH), hydrogen atoms (H) and hydrated electrons (eaq) (Fricke and Morse, 1927). In the presence of oxygen, eaq and H are transformed quantitatively into superoxide anions (O2) and perhydroxyl radicals (HO2), respectively. HO2 is the acidic form of O2 (pKa 5.7) (Spinks and Woods, 1990). The radiolytic yields of O2 and OH formation under our conditions were 3.4 107 and 2.8 107 mol/J. Gamma irradiations were carried out using a 60Co Gamma cell 220 (Atomic Energy of Canada Ltd.) as previously described (Khalil et al., 1998a,b). The dose rate was 0.13 Gy/s as determined by Fricke dosimetry (Fricke and Morse, 1927). Doses of radiation used were 20, 40, 80, 120, 160, and 200 Gy. LDL oxidation induced by free radicals produced by gamma-radiolysis was monitored as follows: CD formation, irradiated lipoproteins (LDL and HDL) (100 mg/ml) were continuously monitored at 234 nm (Khalil et al., 1998a,b) to detect the formation of CD as previously described (Khalil et al., 2000). TBARS formation was assayed as described by Yagi (1976), but without precipitation with phosphotungstic acid (Bonnefont-
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Rousselot et al., 1995). The coefficient of variation of this assay is less than 10% (Khalil et al., 2000). TBARS concentrations were calculated as malondialdehyde (MDA) equivalents using the MDA standard curve. MDA was generated by the hydrolysis of 1,1,3,3tetraethoxypropane. 2.8. Data analysis Given the small size of our experimental sample, nonparametric procedures were used. For between-group comparisons, continuous variables were analyzed using the Kruskall–Wallis test for independant samples. Fisher’s exact test was used to compare the groups on categorical variables (gender). Within-group comparisons were made with the Wilcoxon signed rank test. Data on LDL and HDL irradiation curves were analyzed using an ANOVA method with repeated measurement. All data are presented as the mean S.D. The alpha level of statistical tests was set at 0.05 unless specified otherwise.
69
Table 1 Population description Parameter/groups
Placebo
0.5 g Vit-C
1.0 g Vit-C
N Age (years) Female/male Duration diabetes (years) Body mass index (kg/m2) Fasting glycemia (mmol/l) HbA1c (%) Total cholesterol (mmol/l) LDL (mmol/l) HDL (mmol/l) Triglycerides (mmol/l) Creatinine (mmol/l) Dietary Vit-C (mg/day)
12 71 4 10/2 7.7 8.9 28.9 1.9 8.4 1.6 7.3 0.8 5.22 0.92 2.93 0.84 1.08 0.34 2.67 0.81 94 15 86 46
12 72 5 9/3 10.5 11.8 29.8 3.7 8.2 2.2 7.4 0.9 4.64 0.93 2.72 0.82 1.1 0.34 2.12 1.26 90 36 89 60
12 72 4 9/3 10.2 9.4 29.5 5.3 8.3 2.9 7.4 0.8 4.63 0.76 2.51 0.80 1.02 0.3 2.43 0.83 87 18 78 41
Daily dose Glibenclamide (mg/day) Metformin (g/day) Insulin (units/day)
13.6 6.9 (7)a 1.5 0.4 (5) 84 (1)
8.8 5.4 (6) 1.2 0.6 (9) 30 (1)
11.8 7.4 (11) 1.5 0.6 (9) 57 38 (2)
a Number between parenthesis = number of patients/group under this medication.
3. Results 3.4. Llipid peroxidation markers and susceptibility of LDL and HDL to oxidation induced by gamma-radiolysis
3.1. Baseline data Table 1 shows the demographic characteristics, the biochemical parameters and the daily dietary intake of Vit-C of the 36 subjects who participated in our study. Considering the parameters reported in Table 1, no difference was observed between groups at baseline and within groups at week 12. 3.2. Vit-E content of LDL and HDL Vit-E content of LDL and HDL was similar between groups at baseline. Vit-E content of LDL was significantly increased in subjects treated with 1 g of Vit-C at week 12 (p < 0.05) (Table 2). No significant change in Vit-E content of LDL was observed in subjects under placebo or Vit-C 0.5 g. Vit-E content of HDL was similar between groups at baseline. Vit-C administration at any doses had no effect on the Vit-E content of HDL. 3.3. Cellular level of Vit-C and glutathione At week 12, cellular levels of Vit-C were significantly higher in the 0.5 and 1.0 g groups compared to placebo (p < 0.05). However, the within-group comparisons demonstrated a significant increase of Vit-C only in the 1.0 g group between baseline and week 12 (p < 0.01). For cellular levels of GSH, a significant increase was observed in the 0.5 and the 1.0 groups compared to baseline (p < 0.05) and compared to placebo (p < 0.01) (Table 3). No change was observed for cellular levels of GSSG in the three groups before and after Vit-C supplementation.
Despite an increased content in Vit-E in LDL particles of patients taking 1.0 g of Vit-C (Table 2), no significant change in basal levels of markers of lipid peroxidation (TBARS and CD) in LDL and HDL was observed between baseline and week 12 in the three experimental groups (Table 4). Curves of TBARS and CD generated by gamma-radiolysis of LDL and HDL were analyzed in regard to group and dose/group effects at baseline and at week 12 with an ANOVA method (Table 5). At week 12, no significant difference was detected for these markers in the two Vit-C groups compared to placebo. 4. Discussion Previous studies have suggested that T2 DM is a disease in which reactive oxygen species (ROS) are involved in the pathogenesis of complications (Brownlee, 2001). An epidemiologic study has observed that plasma levels of the antioxidant Vit-C were inversely correlated with HbA1c in diabetic patients (Will et al., 1999). However, in middle-aged subjects with T2 DM, this abnormality is only partially corrected with an oral supplementation of 1 g daily Vit-C (Sinclair et al., 1991). We recently documented that in elderly patients with type 2 DM, hyperglycemia following a meal induces a depletion of Vit-C and an accumulation of GSSG at cellular level (Tessier et al., 1999a). It has been observed that LDL from subjects with T2 DM are more susceptible to oxidation (Yoshida et al., 1997). To normal subjects and to patients with T2 DM, the supplementation of Vit-E
Table 2 Vit-E content of LDL and HDL Group
Placebo
0.5 g Vit-C
1.0 g Vit-C
LDL (mM) Baseline Week 12
1.27 0.51 (7.0 2.8)a 1.48 0.40 (8.2 2.2)
1.31 0.54 (7.2 3.0) 1.57 0.47 (8.7 2.6)
1.23 0.41 (6.8 2.3) 1.98 0.38b,c (10.9 2.1)
HDL (mM) Baseline Week 12
0.55 0.19 (3.1 1.1) 0.60 0.33 (3.3 1.8)
0.52 0.18 (2.9 1.0) 0.60 0.28 (3.3 1.5)
0.46 0.17 (2.6 1.0) 0.63 0.20 (3.5 1.1)
a b c
In parenthesis: number of Vit-E molecules/LDL or HDL particle. p < 0.05 vs. baseline. p < 0.05 vs. placebo group.
D.M. Tessier et al. / Archives of Gerontology and Geriatrics 48 (2009) 67–72
70 Table 3 Cellular levels of Vit-E and glutathione Groups
0.5 g Vit-C
1.0 g Vit-C
Vit-C (nmol/mg prot) Baseline 2.42 2.01 Week 12 2.72 1.88
3.36 1.99 5.39 1.90a
1.45 1.20 5.66 2.00a,b
GSH (nmol/mg prot) Baseline 0.50 0.70 Week 12 0.33 0. 27
0.36 0.23 0.60 0.26c,d
0.37 0.40 0.93 0.70c,d
GSSG (nmol/mg prot) Baseline 0.18 0.27 Week 12 0.33 0.43
0.34 0.28 0.36 0.14
0.26 0.28 0.29 0.27
a b c d
Placebo
p < 0.01 vs. placebo. p < 0.01 vs. baseline. p < 0.01 vs. placebo. p < 0.05 vs. baseline.
protected their LDL against in vitro peroxidation (Jialal et al., 1995; Upritchard et al., 2000). It has been proposed that recycling of Vit-E is dependent on Vit-C (Chan, 1993). In vitro studies observed that the addition of 40–60 mM of Vit-C can prevent initiation and terminate lipid peroxidation in LDL by recycling Vit-E (Retsky and Frei, 1995; Tribble et al., 1995). Vit-E is distributed in the core and in the envelope of the LDL (Cazzola et al., 1999). It has been proposed that the hydrophilic properties of Vit-C facilitate their localization at the interface of the lipid bilayers in membranes, Table 4 TBARS and CD contents of LDL and HDL Group
Placebo
0.5 g Vit-C
1.0 g
LDL Baseline Week 12
0.13 0.19 0.11 0.14
0.13 0.17 0.27 0.20
0.27 0.19 0.17 0.19
HDL Baseline Week 12
0.36 0.75 0.18 0.25
0.13 0.16 0.27 0.29
0.11 0.13 0.11 0.16
0.27 0.12 0.24 0.09
0.23 0.05 0.23 0.11
0.20 0.06 0.27 0.11
0.15 0.11 0.16 0.08
0.14 0.04 0.13 0.05
0.12 0.06 0.21 0.11
TBARS (mmol/l)
CD (OD at 234 nm) LDL Baseline Week 12 HDL Baseline Week 12
Table 5 ANOVA table TBARS and CD contents of LDL and HDL under gamma-radiolysis Effect
Group
Dose group
TBARS (mmol/l) LDL Baseline Week 12
0.39 0.37
0.44 0.70
HDL Baseline Week 12
0.61 0.50
0.50 0.69
0.90 0.03
0.71 0.16
0.46 0.10
0.67 0.41
CD (OD at 234 nm) LDL Baseline Week 12 HDL Baseline Week 12
thereby suggesting two advantages: (i) effective inhibition of attack by free radicals in the aqueous phase, and (ii) effective repair of lipophilic antioxidants (Laranjinha and Cadenas, 1999). Our observation that the in vivo administration of 1 g of Vit-C daily increases Vit-E content of LDL adds one more argument that Vit-C seems to positively modulate Vit-E levels by being moderately protective for Vit-E. In an in vitro model comparing the susceptibility to oxidation of dense versus buoyant LDL, addition of Vit-C resulted in a significant prolongation of the lag time for the 50% depletion of Vit-E in the dense LDL compared to the buoyant ones (Tribble et al., 1995). This result is especially pertinent in that patients with T2 DM have smaller LDL which are more susceptible to in vitro oxidation (Yoshida et al., 1997). Despite the in vitro effect of Vit-C on susceptibility of LDL to oxidation, a clinical study in middleaged patients with type 2 DM failed to show any effect of an oral daily dose of 0.5 g of Vit-C on the susceptibility of LDL to oxidation (Upritchard et al., 2000). Our results seem to confirm the data obtained in this latter study. However, regarding our results concerning the lack of change of LDL susceptibility to oxidation by gamma-radiolysis, we should take into account the magnitude of the effect of oral supplementation in Vit-C in regard to the Vit-E content of LDL. In a dose–response study with Vit-E, the Vit-E content of LDL increased by as much as 175% with the 1200 IU/day dosage (Jialal et al., 1995). In our study, the size of the effect was +61% for Vit-E content in LDL of patients taking 1 g daily of Vit-C. There are no data whether higher doses of Vit-C administration would increase more the LDL content of this vitamin. This needs further studies. Moreover, it should be noted that in the process of LDL isolation, ultracentrifugation removes the plasma containing the antioxidants such as Vit-C and glutathione that are usually present in the biological environment of the lipid particles. HDL is involved in the protection of LDL against lipid peroxidation via the enzymes paraoxonase and platelet acetylating factor acetylhydrolase (Watson et al., 1995; Hedrick et al., 2000; Berrougui et al., 2007), and glycation diminishes paraoxonase activity (Mackness et al., 1998). We observed that products of lipid peroxidation can be measured in HDL of patients with type 2 DM (Tessier et al., 1999b). An epidemilogical study observed that each 1 mg/dl increase in serum Vit-C was independently associated with an increase of 2–3 mg/dl in HDL-C level (Simon and Hudes, 1998), but no clinical study yet, including our present one, documented that oral supplementation of Vit-C could diminish basal levels of lipid peroxidation products present in HDL and susceptibility of this particle to peroxidation. GSH, a tripeptide containing a sulfhydryl group, plays a key role in detoxification of ROS. Observational studies demonstrated a correlation of Vit-C and glutathione in normal elderly subjects (Lenton et al., 2000). It has been suggested in the literature that VitC and glutathione function as a redox couple (Winkler et al., 1994). When Vit-C is oxidized to dehydroascorbate (DHAA), a glutathione-dependent DHAA-reductase activity may be important in the physiologic recycling of Vit-C (Vethanayagan et al., 1999). Under experimental oxidative stress with hydrogen peroxide, VitC may preferentially react with the ROS and by doing so, preserves the reserve in glutathione (Sturgeon et al., 1998). One animal study demonstrated that increasing dietary content in Vit-E resulted in a gradual increase in the GSH/GSSG ratio and supplementation with 100 IU Vit-E/day in children with type 1 diabetes increased erythrocyte level of glutathione (Jain et al., 2000). These observations suggest a protecting role of Vit-E on GSH and may be proposed as elements to explain the increase in the cellular levels of GSH that we observed in our present study. In addition, GSH regenerates Vit-E in vitro with the aid of protein catalysis (McCay, 1985; Chan, 1993). Our results demonstrating that Vit-C
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supplementation can increase the cellular Vit-C and GSH levels suggest that it is possible to modulate the neutrophils and possibly, other cell antioxidant content in a context of oxidative stress such as T2 DM. This could confer a better functioning and a better innate immune defense in these cells. Altogether these data show that it exists an interacting network between various antioxidants modulating the content and activity of each other. Recent studies have suggested additional interactions between Vit-C and T2 DM. In an observational study, an inverse relationship between Vit-C levels and markers of DNA damages (Choi et al., 2005) has been demonstrated. In human mononuclear cells involved in the atherogenic process, Vit-C down regulates the genes associated with proliferation (Kaul and Baba, 2005). In patients with T2 DM and coronary artery disease, Vit-C supplementation for 4 weeks increases forearm blood flow (Tousoulis et al., 2003). The mechanism of this increment in blood flow seems to be independent of nitric oxide (Ajay and Mustafa, 2006). In summary, oral supplementation with Vit-C in elderly patients with T2 DM, increases cellular reserve in antioxidants namely Vit-C and glutathione, and the Vit-E content of LDL in a dose-dependent manner. The lack of effect of Vit-C on lipid peroxidation markers and susceptibility of lipid particles to oxidation is probably due to the relatively small size of the effect of Vit-C on Vit-E. Our study confirms the in vivo interactions between antoxidants but the clinical benefits of these changes remains unclear. We still lack clinical data for clear-cut recommendation for the use of antioxidants in various clinical settings. Nevertheless, studies such as ours contribute for the better understanding of the in vivo effects of Vit-C in special clinical settings such as T2 DM. More controlled clinical trials are needed to fully elucidate the effects of antioxidants including Vit-C on cellular and molecular markers of oxidative stress and on disease pathogenesis and progression. References Ajay, M., Mustafa, M.R., 2006. Effects of ascorbic acid on impaired vascular reactivity in aortas isolated from age-matched hypertensive diabetic rats. Vascul. Pharmacol. 45, 127–144. Berrougui, H., Isabelle, M., Cloutier, M., Grenier, G., Khalil, A., 2007. Age-related impairment of HDL-mediated cholesterol efflux. J. Lipid Res. 48, 328–336. Block, G., 1982. A review of validations of dietary assessment methods. Am. J. Epidemiol. 115, 492–505. Bode, A.M., Rose, R.C., 1999. Analysis of water-soluble antioxidants by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 299, 77–83. Bonnefont-Rousselot, D., Motta, C., Khalil, A., Sola, R., La Ville, A.E., Delattre, J., Gardes-Albert, M., 1995. Physicochemical changes in human high-density lipoproteins (HDL) oxidized by gamma radiolysis-generated oxyradicals. Effect on their cholesterol effluxing capacity. Biochim. Biosphys. Acta 1255, 23–30. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820. Cazzola, R., Cervato, G., Cestaro, B., 1999. Variability in alpha-tocopherol antioxidants activity in the core and surface layers of low- and high-density lipoproteins. J. Nutr. Sci. Vitaminol. 45, 39–48. Chan, A.C., 1993. Partners in defense, vitamin E and vitamin C. Can. J. Physiol. Pharmacol. 71, 725–731. Choi, S.W., Benzie, I.F., Lam, C.S., Chat, S.W., Lam, J., Yiu, C.H., Kwan, J.J., Tang, Y.H., Yeung, G.S., Yeung, V.T., Woo, G.C., Hannigan, B.M., Strain, J.J., 2005. Interrelationships between DNA damage, ascorbic acid and glycaemic control in type 2 diabetes mellitus. Diabet. Med. 22, 1347–1353. De Leenher, A.P., De Bevere, V.O., Cruyl, A.A., Claeys, A.E., 1978. Determination of serum alpha-tocopherol (vitamin E) by high-performance liquid chromatography. Clin. Chem. 24, 585–590. Fortin, C.F., Lesur, O., Fulop, T., 2007. Effects of aging on triggering receptor expressed on myeloid cells (TREM)-1-induced PMN functions. FEBS Lett. 581, 1173–1178. Fricke, H., Morse, S., 1927. The chemical action of Ro¨ntgen rays on dilute ferrosulfate solutions as a measure of dose. Am. J. Roentgen Radiat. Ther. 18, 430–432. Hedrick, C.C., Thorpe, S.R., Fu, M.-X., Harper, C.M., Yoo, J., Kim, S.-M., Wong, H., Peters, A.L., 2000. Glycation impairs high-density lipoprotein function. Diabetologia 43, 312–320. Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214–226.
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