|Year : 2021 | Volume
| Issue : 1 | Page : 16-23
Vitamin C supplementation improves blood pressure and oxidative stress after acute exercise in patients with poorly controlled type 2 diabetes mellitus: A randomized, placebo-controlled, cross-over study
Chongchira Boonthongkaew1, Terdthai Tong-Un2, Yupaporn Kanpetta3, Nisa Chaungchot4, Chanvit Leelayuwat5, Naruemon Leelayuwat6
1 Biomedical Sciences Program, Graduate School; Exercise and Sport Sciences Development and Research Group, Khon Kaen University, Khon Kaen, Thailand
2 Exercise and Sport Sciences Development and Research Group; Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
3 Exercise and Sport Sciences Development and Research Group, Khon Kaen University, Khon Kaen; Sports and Exercise Science Program, Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus, Nong Khai, Thailand
4 Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
5 Centre for Research and Development of Medical Diagnostic Laboratories; Department of Clinical Immunology and Transfusion Sciences, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
6 Exercise and Sport Sciences Development and Research Group; Graduate School, Khon Kaen University, Khon Kaen, Thailand
|Date of Submission||24-Nov-2020|
|Date of Decision||04-Dec-2020|
|Date of Acceptance||08-Dec-2020|
|Date of Web Publication||25-Feb-2021|
Assoc. Prof. Naruemon Leelayuwat
Exercise and Sport Sciences Development and Research Group, Graduate School, Khon Kaen University, Khon Kaen 40002
Source of Support: None, Conflict of Interest: None
This study aimed to assess the effect of Vitamin C on blood pressure (BP), and subsequently on oxidative stress and nitric oxide (NO) release, following the low-intensity exercise in the patients. This study included 24 patients with type 2 diabetes mellitus (T2D) (age, 53 ± 7 years; hemoglobin A1c, 10.1% ± 0.9%) randomized into two 6-week daily arms based on the consumption of either placebo or 1000 mg Vitamin C. The crossover trial occurred after a 6-week washout. Before and after both supplementation arms, all patients performed cycling exercise at 33% of peak oxygen consumption for 20 min. BP was measured before, immediately, and 60 min after the exercise. Blood samples were drawn immediately before and after the exercise to determine plasma ascorbate, malondialdehyde (MDA), F2-isoprostanes (F2-IsoPs), and NO concentrations. Data showed significant lower BP in the Vitamin C arm when compared with the placebo arm (systolic BP [SBP] P < 0.001 at every time point, diastolic BP [DBP] P < 0.001 except at immediately after exercise, P < 0.05). Plasma ascorbate concentration (P < 0.05 at every time point) and plasma NO (at resting P < 0.001, immediately after exercise P < 0.05) were significantly increased in the Vitamin C arm than in the placebo arm. Plasma MDA (P < 0.05 at every time point) and F2-IsoPs (P < 0.05 at every time point) concentrations were significantly lower in the Vitamin C arm than in the placebo arm. In addition, data showed significantly lower SBP (P < 0.001 at every time point), DBP (P < 0.001 except at immediately after exercise P < 0.05), plasma MDA (P < 0.001 at every time point), and F2-IsoPs (P < 0.05 at every time point) at post-supplementation than at pre-supplementation. Besides, there were significantly higher plasma ascorbate (P < 0.05 at every time point) and NO (at rest P < 0.01, immediately after exercise P < 0.05) concentrations at post-supplementation than at pre-supplementation. This is in contrast to the placebo treatment arm which demonstrated no statistical difference in all outcomes throughout the experiment. This study suggests that 6-week Vitamin C supplementation decreased preexercise and postexercise BPs, possibly due to improved oxidative stress and NO release. However, exercise had no effect on any outcome measures.
Keywords: Ascorbic acid, F2-isoprostanes, hyperglycemia, hypertension, malondialdehyde, physical activity
|How to cite this article:|
Boonthongkaew C, Tong-Un T, Kanpetta Y, Chaungchot N, Leelayuwat C, Leelayuwat N. Vitamin C supplementation improves blood pressure and oxidative stress after acute exercise in patients with poorly controlled type 2 diabetes mellitus: A randomized, placebo-controlled, cross-over study. Chin J Physiol 2021;64:16-23
|How to cite this URL:|
Boonthongkaew C, Tong-Un T, Kanpetta Y, Chaungchot N, Leelayuwat C, Leelayuwat N. Vitamin C supplementation improves blood pressure and oxidative stress after acute exercise in patients with poorly controlled type 2 diabetes mellitus: A randomized, placebo-controlled, cross-over study. Chin J Physiol [serial online] 2021 [cited 2022 Jul 1];64:16-23. Available from: https://www.cjphysiology.org/text.asp?2021/64/1/16/310132
| Introduction|| |
Type 2 diabetes mellitus (T2D) is one of the most common metabolic disorders and comorbid conditions in the patients including endothelial dysfunction and cardiovascular diseases, including hypertension (HTN),, resulting in increased morbidity and mortality. T2D patients with poor glycemic control (hemoglobin A1c [HbA1c] >7%) are likely to have more severe impairments.
A single bout of steady-state exercise has been shown to promote postexercise hypotension (PEH). During the exercise, mechanical stress on the arterial wall is increased, leading to an increased release of vasodilators by the endothelium (e.g., nitric oxide [NO], bradykinin, etc.)., This response can attenuate blood pressure (BP) after acute exercise at low, moderate, and high intensity in normotensive individuals. However, the magnitude of this effect seems to decline in T2D patients because of endothelial dysfunction.
A significant factor for the pathogenesis of endothelial dysfunction is the high oxidative stress caused by the imbalance between the excessive formation of free radicals and the limited antioxidant defenses. The oxidative stress results in impaired NO bioavailability, and NO is required for vasodilation. Lipid peroxidation-generated end products, which are the most common biomarkers of oxidative stress, include malondialdehyde (MDA) and F2-isoprostanes (F2-IsoPs)., Therefore, administering antioxidants may help the patients gain benefits from the exercise by improving oxidative stress, thereby leading to improved endothelial function; this improvement could be determined by the increase in PEH.
Although moderate- to high-intensity exercise for at least 30 min/session is recommended for T2D patients, these exercises could result in inflammation and muscle cell damage. Exercise at low intensity for 20 min was used in our study because it is more practical for T2D patients who exhibit reduced exercise tolerance, muscle weakness, and cardiovascular comorbidities. Importantly, exercise at low intensity performed at 30% of peak oxygen consumption (V̇O2peak) also provoked hypotension during the 60-min recovery, which correlates with the hypotension occurring at 50% and 80% of V̇O2peak in normotensive individuals. However, T2D patients might have decreased PEH owing to endothelial dysfunction. The combination of Vitamin C, one of the most powerful antioxidants,, with the low-intensity exercise regimen may improve BP recovery. However, there are no studies exploring the effect of Vitamin C on PEH and subsequent responses of lipid peroxidation and NO release to low-intensity exercise in patients with poorly controlled T2D.
This study, thus, aimed to determine the effects of oral Vitamin C supplementation on PEH and subsequently on lipid peroxidation and NO release following low-intensity exercise in patients with poorly controlled T2D. It is hypothesized that the supplementation of Vitamin C would result in PEH and improved lipid peroxidation and NO release in response to low-intensity exercise in T2D patients.
| Materials and Methods|| |
Ninety-five T2D patients (mean age, 53 ± 7 years) with poor glycemic control were screened at Khon Kaen University from January 2014 to October 2016. Twenty-four were considered eligible to participate in this study (20 women and four men). Patients were recruited through personal contacts, posters, and advertisements. The inclusion criteria were age 45–60 years, diagnosis of T2D at least 12 months prior to the study, glycated HbA1c level >7%, oral hypoglycemic drug treatment, normal lipid profile or dyslipidemia with or without lipid-lowering drugs, systolic BP (SBP) ≤140 mmHg or diastolic BP (DBP) ≤90 mmHg, antihypertensive drug treatment at a similar dose throughout the study for maintaining a BP of 140/90 mmHg, sedentary lifestyle and not having participated in any regular exercise program (<3 times/week) for at least 6 months before the study, and residence in Khon Kaen Province, Thailand. BP was measured using an automatic sphygmomanometer (UA-767 Plus, Abingdon, Oxfordshire, UK; calibrated annually). The exclusion criteria were any conditions that would restrict the ability of the patient to participate in the study, such as receiving insulin injection, orthopedic problems, neuromuscular disorders, liver and/or kidney diseases, and chronic infections. Patients were also excluded if their treatment plan or medication were changed during the study. Total body composition, lean body mass, and body fat mass were measured using dual-energy X-ray absorptiometry (Lunar Prodigy whole-body scanner, GE Healthcare, Chicago, IL, USA). Body mass and height were measured using a stadiometer (DETECTO, St. Webb City, MO, USA). Patients were asked to maintain their normal daily physical activity and dietary habits during the study period. They were requested to record their physical activity and food intake for 3 days each week (2 weekdays and 1 weekend). The records were used for estimating energy intake and expenditure. Energy intake was analyzed using the INMUCAL program (INMUCAL software, Mahidol University, Salaya campus, Nakorn Pathom, Thailand). All patients provided written and verbal informed consent. The study was approved by the human ethical committee of the institution according to the 1964 Declaration of Helsinki, and the study was registered as a clinical trial on the Thai Clinical Trials website. Trials Registry Number: TCTR20180226002, registered date: October 13, 2013.
Twenty-four patients (considering a 20% dropout rate) were recruited to this study according to statistical calculation requirements using the WinPepi program. Mullan et al. reported a statistically significant decrease of SBP 4.4 mmHg due to the effects of Vitamin C for patients with T2D in their study (P < 0.05, α = 0.05, β = 0.80 and power = 0.80).
Study design and randomization
This prospective, double-blind, placebo-controlled cross-over study was conducted at Khon Kaen University. The randomized treatment allocation sequence was generated using random number tables, which were operated by research assistants, resulting in an equal proportion of patients in the experimental (Vitamin C) and control (placebo) supplementation arms [Figure 1]. Before allocation by another research assistant, each number was kept in a sealed envelope. Neither the researchers nor the patients was aware of the allocation until the study was completed.
|Figure 1: Protocol of this study. (a) Immediately before exercise, (b) immediately after exercise, (c) 60 min after exercise. T2D: Type 2 diabetes mellitus, V̇O2peak: Peak oxygen consumption, EXS: Exercise.|
Click here to view
Vitamin C and placebo preparation
Both Vitamin C (Blackmores Bio C 1000 mg) and placebo supplements were provided by Blackmores Institute (Warriewood, NSW, Australia; Reg. No. 2c 70/40). The Vitamin C supplement consisted of 1000 mg ascorbic acid. Purity, composition, and toxicity were assessed by the company to comply with the ethical requirements of the study. The placebo supplement consisted of 1000 mg of Daucus carota dry root. The placebo had the same color, shape, size, odor, and packaging as the Vitamin C supplement. There were 42 tablets of supplement in each dark glass bottle.
Patients visited the laboratory on five different occasions [Figure 1]. On the first visit, V̇O2peak was tested for setting a workload for the 20-min exercise test on a cycle ergometer. One week later, all patients were randomly and equally divided into two supplementation arms (to prevent order effect): patients were given either a 1000 mg placebo supplement or a 1000 mg Vitamin C supplement daily for 6 weeks, with a 6-week wash-out period between the study arms. On the day before the first day and after the last day of each supplementation phase, all patients performed 20-min cycling at the workload equivalent to a 30% V̇O2peak. Immediately before and after all exercise tests, venous blood samples were drawn; BP was also measured immediately before, and immediately, and 60 min after the exercise. Blood samples were used for determining plasma ascorbate, MDA, F2-IsoPs, NO (nitrite/nitrate) concentrations. All visits during the study started at 8 AM. The room temperature was 25°C, and humidity was 57% ± 7% (mean ± standard deviation) during the testing.
Blood sample collection
Blood samples (14 mL each) were collected from the antecubital vein into two tubes. A 12-mL ethylenediaminetetraacetic acid (EDTA) tube was used for measuring MDA, F2-IsoPs, and NO levels in plasma. The other 2-mL EDTA tube was used for measuring plasma ascorbate concentrations. The EDTA tube for plasma ascorbate assay was wrapped in aluminum foil and placed in an ice bath prior to centrifugation. All tubes were centrifuged at 3000g for 10 min at 4°C, and the plasma was collected. Aliquots of plasma were frozen immediately and stored at −80°C before the analysis. Samples for NO assay were filtered using a 10-kDa molecular weight cutoff ultrafilter before assay for reducing protein interference and turbidity.
Outcome variables measurements
Blood pressure measurement
An automatic sphygmomanometer (UA-767 Plus, Abingdon, Oxfordshire, UK; calibrated annually) was used for measuring SBP and DBP with the cuff wrapped around the upper right arm of the patient at the brachial artery. Triplicate measurements were acquired under standardized conditions.
Plasma ascorbate measurement
Plasma ascorbate concentration was measured using Zhang's method. Briefly, 100 μL of each deproteinized plasma sample was mixed with 100 μL of ferric chloride (Panreac AppliChem ITW companies, Darmstadt, Germany) and 100 μL of potassium ferricyanide (Ajax Finechem Pty Ltd., Taren Point, NSW, Australia). Then 2200 μL of distilled water was added for adjusting the total volume of solution to 2500 μL. The solution was incubated at 20°C in an ice bath for 30 min. After the incubation, the mixture was centrifuged at 2059 g for 10 min. The supernatant was measured at 735 nm using a spectrophotometer. During the entire process, the samples were always incubated in ice and never exposed to light.
Plasma malondialdehyde measurement
Plasma MDA concentrations were measured using Draper's method. Briefly, 150 μL of plasma was mixed with 125 μL of 10% trichloroacetic acid (Aldrich Chemistry, Darmstadt, Germany), 125 μL of 5 mM EDTA (Sigma Life Science, Darmstadt, Germany), 125 μL of 8% sodium dodecyl sulfate (Sigma Life Science, Darmstadt, Germany), and 10 μL of 0.5 μg/mL butylated hydroxytoluene (Aldrich Chemistry, Darmstadt, Germany). Then, the solution was incubated at the room temperature for 10 min. Next, 125 μL of 0.6% thiobarbituric acid (Sigma-Aldrich, Steinheim, Germany) was added to the solution, and the mixture was boiled in a water bath for 10 min. After cooling, the mixture was centrifuged at 2095 g for 10 min. The absorbance of the supernatant was then measured at 532 nm using a spectrophotometer.
Plasma F2-isoprostanes measurement
F2-IsoPs was measured using a direct 8-iso-prostaglandin F2α enzyme immunoassay kit (EIA, 8-iso-PGF2α kit, Cayman Chemical Company, Ann Arbor, MI, USA). This assay is based on the competition between 8-isoprostane and 8-isoprostane-acetylcholinesterase (AChE) conjugate (8-isoprostane tracer) for a limited number of 8-isoprostane-specific rabbit antibody binding sites. The concentration of 8-isoprostane tracer was constant, while the concentration of 8-isoprostane varied, and the amount of 8-isoprostane tracer that can bind to the rabbit antibody is inversely proportional to the concentration of 8-isoprostane in the well. The rabbit antibody-8-isoprostane (either free or tracer) complex binds to the rabbit IgG mouse monoclonal antibody that has been previously attached to the well. The plate was then washed to remove any unbound reagents, and Ellman's reagent (with the substrate and AChE) was added to the well. The product of this enzymatic reaction has a distinct yellow color, which was detected at 412 nm using a spectrophotometer.
Plasma nitric oxide (nitrite/nitrate) measurement
Owing to its extremely short half-life, the direct measurement of NO production cannot be assessed by most detection systems. Instead, total NO is measured indirectly using the nitrite and nitrate ratio. Thus, in this case, plasma nitrite/nitrate concentration was measured using a colorimetric assay kit (OxiSelect™ Nitric Oxide (Nitrite/Nitrate) Assay Kit, Cell Biolab, Inc., San Diego, CA, USA). All reagents were maintained at 4°C during assay preparation. A freshly prepared standard curve was used each time the assay was performed. The nitrate in the sample is converted to nitrite by the nitrate reductase enzyme. Total nitrite was then detected with the Griess reagent as a colored azo dye product (absorbance at 540 nm).
Data were analyzed using the SPSS Statistics software package for Windows, version 20 (IBM Corp., Armonk, NY, USA). Normal distribution of the data was tested using the Shapiro–Wilk Test. The differences within and between supplementations were tested using the repeated-measures analysis of variance. When a significant difference was observed, a post hoc analysis using the Bonferroni adjustment was performed. All differences were considered significant at a P < 0.05. All data were expressed as mean ± standard error of the mean (standard error), except where stated otherwise. Cohen's d was calculated for measuring effect size.
| Results|| |
Twenty-four patients (20 women and four men) were included in the study and 20 patients (16 women and four men) (83% of total patients) completed the study. Patients were overweight to obese without any differences between pre- and post-supplementation in both the supplementation arms in terms of anthropometric and physiological characteristics. All of them had low physical fitness. Furthermore, there were no significant differences in blood chemistry and energy intake and energy expenditure in both the arms (P > 0.05) [Table 1]. Several patients were receiving hypoglycemic, anti-hypertensive, and lipid-lowering therapies, but none of them required insulin injections. All patients maintained their medication plans throughout the study. No patient in this study experienced any adverse effects for the duration of the study.
|Table 1: Baseline biochemical characteristics and energy intake and expenditure of T2D patients|
Click here to view
Within Vitamin C-arm data showed significantly lower SBP and DBP at rest before exercise (SBP; −12.8 ± 1.4 mmHg, 95% confidence interval [CI], −17.1 – [−8.5], P < 0.001, d = 1.63, DBP; −8.9 ± 1.8 mmHg, 95% CI, −14.5 – [−3.3], P < 0.001, d = 1.02) at post-supplementation than at pre-supplementation. The data also showed significantly lower SBP and DBP immediately after exercise (SBP; −11.4 ± 1.3 mmHg, 95% CI, −15.4 – [−7.3], P < 0.001, d = 1.53, DBP; −6.8 ± 2.0 mmHg, 95% CI, −12.9 – [−0.6], P < 0.05, d = 0.82). Besides, the data showed lower SBP and DBP 60 min after exercise (SBP; −12.5 ± 1.4 mmHg, 95% CI, −16.7 – [−8.2], P < 0.001, d = 1.64, DBP; −8.9 ± 1.8 mmHg 95% CI, −14.5 – [−3.2], P < 0.001, d = 1.03) [Figure 2]a and [Figure 2]b. Whereas the statistical differences were not observed in the placebo arm.
|Figure 2: Systolic blood pressure (a) and diastolic blood pressure (b) at rest (T0), immediately (T1), and 60 min (T2) after exercise at pre- and post-supplementation of both arms. The data represent mean ± SE (n = 20, men = 4, women = 16). SE: Standard error. *Significantly different from pre-supplementation in the same supplementation arm (P < 0.001), §Significantly different from placebo supplementation arm in the same period of time (P < 0.001).|
Click here to view
This significant decrease in BP was conserved after the Vitamin C supplementation when compared with after the placebo supplementation at rest before exercise (SBP; −10.1 ± 2.4 mmHg, 95% CI, −14.9 – [−5.2], P < 0.001, d = 1.34, DBP; −8.6 ± 1.9 mmHg, 95% CI, −12.3 – [−4.9], P < 0.001, d = 1.47). Furthermore, the significant difference was observed immediately after exercise (SBP; −9.4 ± 2.3 mmHg, 95% CI, −14.0 – [−4.7], P < 0.001, d = 1.28, DBP; -5.3 ± 1.9 mmHg, 95% CI, −9.0 – [−1.5], P < 0.05, d = 0.89). Sixty min after exercise the significant decrease in BP was also found (SBP; −9.9 ± 2.4 mmHg, 95% CI, −14.6 – [−5.1], P < 0.001, d = 1.33, DBP; −8.7 ± 1.8 mmHg, 95% CI, −12.4 – [−5.0], P < 0.001, d = 1.51) [Figure 2]a and [Figure 2]b. However, the statistical differences of BP between resting before and within 60 min after exercise were not observed in both Vitamin C and placebo arm.
Plasma ascorbate concentration
Within Vitamin C-arm data showed significantly higher plasma ascorbate concentrations at rest before and immediately after exercise at post-supplementation than at pre-supplementation (at rest; 26.3 ± 4.6 μmol/L, 95% CI: 13.6–38.9, P < 0.001, d = 0.78, immediately after exercise; 18.7 ± 5.3 μmol/L, 95% CI: 4.0–33.5, P < 0.05, d = 0.56) without any difference in the placebo arm. Plasma ascorbate concentration significantly higher at rest before and immediately after low-intensity exercise at post-Vitamin C supplementation than at post-placebo supplementation (at rest, 22.0 ± 9.9 μmol/L, 95% CI: 2.0–42.0, P < 0.05, d = 0.70, immediately after exercise; 20.9 ± 9.9 μmol/L, 95% CI: 0.9–41.0, P < 0.05, d = 0.79).
Plasma malondialdehyde concentration
Within Vitamin C-arm data showed significantly lower plasma MDA concentrations at rest immediately before and after exercise at post-supplementation than at pre-supplementation (at rest; −5.0 ± 1.1 μmol/L, 95% CI: −8.0 – (−2.0), P < 0.001, d = 0.86, immediately after exercise; −6.6 ± 1.5 mmol/L, 95% CI: −10.9 – (−2.4), P < 0.05, d = 1.33). This is in contrast to the placebo treatment arm which demonstrated no statistical difference in plasma MDA concentration [Figure 3]a. Plasma MDA concentration was also significantly lower at rest before and immediately after low-intensity exercise at post-Vitamin C arm than at post-placebo arm (at rest, −5.3 ± 1.9 μmol/L, 95% CI: −9.2 – (−1.4), P < 0.05, d = 0.86, immediately after exercise; −5.9 ± 1.5 mmol/L, 95% CI: −8.4 – (−2.4), P < 0.05, d = 1.12) [Figure 3]a.
|Figure 3: Plasma malondialdehyde (a), F2-Isoprostanes (b), and nitric oxide (c) concentrations at rest and immediately after EXS at pre-supplementation and post-supplementation of both arms. The data represent mean ± SE (n = 20, men = 4, women = 16). EXS: Exercise, SE: Standard error. *Significantly different from pre-supplementation in the same supplementation arm (P < 0.001), †Significantly different from pre-supplementation in the same supplementation arm (P < 0.05), §Significantly different from placebo supplementation arm in the same period of time (P < 0.001), ‡Significantly different from placebo supplementation arm in the same period of time (P < 0.05).|
Click here to view
Plasma F2-isoprostanes concentration
Within Vitamin C-arm data showed a significant lower plasma F2-IsoPs concentrations at rest immediately before and after exercise at post-supplementation than at pre-supplementation (at rest; −4.6 ± 1.2 ng/L, 95% CI: −8.0 – [−1.2], P < 0.05, d = 0.57, immediately after exercise; −7.5 ± 1.5 ng/L, 95% CI: −13.5 – [−1.4], P < 0.05, d = 0.58). Whereas the statistical difference was not observed in the placebo arm [Figure 3]b. Plasma F2-IsoPs concentration at rest before and immediately after exercise also demonstrated significant lower at post-Vitamin C supplementation when compared with a post-placebo supplementation (at rest; −10.1 ± 4.3 ng/L, 95% CI: −18.8 – (−1.5), P < 0.05, d = 0.77, immediately after exercise; −10.6 ± 4.3 ng/L, 95% CI: −19.4 – (−1.8), P < 0.05, d = 0.69) [Figure 3]b.
Plasma nitric oxide concentration
Within Vitamin C-arm data showed that Vitamin C supplementation resulted in a significant higher plasma NO concentrations at rest before and immediately after exercise at post-supplementation than at pre-supplementation (at rest; 54.4 ± 9.9 μmol/L, 95% CI: 26.8–81.9, P < 0.001, d = 1.2, immediately after exercise; 42.1 ± 10.5 μmol/L, 95% CI: 12.9–71.2, P < 0.05, d = 0.89). Whereas placebo had no effect on NO concentration at rest before and immediately after low-intensity exercise [Figure 3]c. Plasma NO concentration was also significantly greater at rest before and immediately after low-intensity exercise at post-Vitamin C supplementation than at post-placebo supplementation (at rest; 59.4 ± 13.9 μmol/L, 95% CI: 31.3–87.5, P < 0.001, d = 1.35, immediately after exercise; 40.8 ± 14.2 μmol/L, 95% CI: 12.1–69.5, P < 0.05, d = 0.99) [Figure 3]c.
| Discussion|| |
This is the first study to report the impact of Vitamin C supplementation on lowering SBP and DBP before and within 60 min after the exercise when compared with placebo supplementation. Besides, we found the Vitamin C supplementation resulted in lower lipid peroxidation and an increase in NO concentration in response to low-intensity exercise in T2D patients with poor glycemic control. This may play a role in the antihypertensive effect of Vitamin C supplementation.
We hypothesized that supplementation of Vitamin C would result in PEH and improve lipid peroxidation and NO release in response to low-intensity exercise in the T2D patients with poor glycemic control. The absence of PEH does not support our initial hypothesis. Unfortunately, no previous studies have revealed any effect of Vitamin C supplementation on BP during recovery after a single session of low-intensity exercise in T2D patients. Nonetheless, based on higher plasma ascorbate concentrations immediately before and after the exercise in the Vitamin C arm in our study, some beneficial effect of oral Vitamin C supplementation on health was suggested. According to the inverse association between plasma ascorbate concentration and the risk of stroke among hypertensive men and between plasma ascorbate concentration and BP in healthy young adults, the increased plasma ascorbate concentration and decreased BP at rest and immediately after exercise in our study may imply the reduction in the risk of stroke in T2D patients. Thus, Vitamin C supplementation and exercise are important for preventing or treating HTN in T2D patients. Besides, we expected that T2D patients with uncontrolled high BP may experience a lower drop in post-exercise BP than normotensive patients. A further study investigating the effect of Vitamin C on post-exercise BP in T2D patients with high BP should be warranted.
Unexpectedly, we did not find BP drop below baseline after the exercise (PEH) in both the supplementation arms. However, BP was lower throughout the experimental session in the Vitamin C arm than in the placebo arm. It may show the effect of oral Vitamin C on antihypertension without any effect from the exercise. Along with lower BP, the higher NO concentration throughout the experimental session in the Vitamin C arm than in the placebo arm may show the effect of oral Vitamin C on improving endothelial function. Theoretically, exercise has been shown to increase mechanical stress on the walls of the arteries, leading to increase NO release and vasodilation. PEH response to exercise itself can be vigorous depending on the effort. Actually, exercise at low intensity (30%V̇O2peak) for 45 min was shown to promote PEH during the first hour recovery. However, the participants in the previous study were normotensive and non-diabetic adults. Participants in this study were T2D patients who have endothelial dysfunction leading to a resistance to exercise-induced BP reduction. Therefore, the lack of PEH in this study may be due to either vascular dysfunction itself or inadequate exercise intensity or duration.
It may be worth noting that although all patients in our study had no PEH, they had SBP and DBP within normal ranges throughout the experimental session. Eight of them had HTN and took antihypertensive drug (except on the exercise day), but the antihypertensive effect promoted by Vitamin C supplementation was observed in all patients. Therefore, the combination of Vitamin C and the acute exercise regimen in our study seemed to be safe for patients.
Furthermore, we confirmed our secondary hypothesis that 1000 mg Vitamin C has a beneficial effect on improving lipid peroxidation and NO concentrations in patients with poorly controlled T2D. The result indicating the decrease in lipid peroxidation concentration resulting from Vitamin C supplementation in our study agrees with that reported in previous studies. Those studies have reported that 3–4 months supplementation with 1000 mg Vitamin C decreases resting plasma MDA and F2-IsoPs concentrations in T2D patients. Vitamin C donates an electron to the lipid radical for terminating lipid peroxidation and inhibiting second end product generation, that is MDA and F2-IsoPs. Moreover, Vitamin C generates other antioxidants such as superoxide dismutase and Vitamin E. These antioxidants could work together to further scavenge reactive oxygen species. However, a recent review reported that Vitamin C did not cause a reduction in MDA. The reason for the lack of MDA reduction may be either the dose or duration of supplementation.,, The doses at 200 and 800 mg/day of Vitamin C supplementation may be too low to have impact on the lipid peroxidation marker. However, the doses at 1500–3000 mg/day with duration of 2–3 weeks may be not efficient enough to improve oxidative stress., The doses may be very high but the supplementation duration seems to be too short. Unfortunately, to date, there has been no report about the effects of Vitamin C on low-intensity exercise-induced MDA and F2-IsoPs concentrations in T2D patients with poor glycemic control.
The results of this study may provide one of potential mechanisms of beneficial effect of Vitamin C on BP. It could be the reduction in oxidative stress because oxygen free radicals may be either the causes or consequences of hypertension., The former may contribute to inactivating endothelium-derived NO and thus attenuating this important vasodilator mechanism. Therefore, the Vitamin C-induced reduction in oxidative stress (indicated by a reduction in MDA and F2 IsoPs levels in this study) may prevent NO from free radical-induced degradation. Besides, previous studies have reported that physiological concentrations of Vitamin C-enhanced synthesis of NO in the human umbilical vein and coronary artery endothelial cells and porcine aortic endothelial cells. To synthesize NO, vitamin C increases intracellular levels of tetrahydrobiopterin, an essential cofactor for endothelial NO synthase activity., This evidence strongly indicates that Vitamin C plays an important role in NO formation. Furthermore, Vitamin C had a marked stimulatory effect on the biological activity of NO, indicated by cGMP levels. The evidence may confirm the effect of oral Vitamin C supplementation on lipid peroxidation and NO release, which protect endothelial function. It has been known that lipophilic derivatives of ascorbic acid contributed to protective effect on lipid peroxidation-induced endothelial injury. This effect then decreases BP at rest before and immediately after a single low-intensity exercise session in our study.
One might suspect about the effect of gender on our outcomes since several participants were women (16 women: 4 men). High levels of estrogen have been shown to have antioxidant effects by inhibiting DNA oxidation and stimulating NO production. Although we did not measure estrogen concentration, all except one woman in this study were menopausal. The estrogen concentrations have been shown to be similar in men and menopausal women. Therefore, gender seems to have no effect on the outcomes of our study. Our results can be applied to both men and menopausal women with T2D. Furthermore, we confirm that the 6-week washout was adequate. According to two previous studies reporting that the average half-life of ascorbic acid in adult human is about 10–20 days; therefore, the 42 days of the 6-week washout is considered to be adequate.,
A limitation of our study is that we did not explore other potential mechanisms explaining the hypotensive effect of oral Vitamin C in T2D patients. The regulation of BP is affected by the mechanisms such as sympathetic nervous system activity, the renin-angiotensin-aldosterone system, and the NO pathway. The NO pathway includes the xanthine oxidase pathway and the nicotinamide adenine dinucleotide phosphate oxidases pathway. The former produces the potent hydroxyl radical, and the latter produces superoxide and hydrogen peroxide. Therefore, studies on the measurements of these mechanisms in response to the effect of long-term Vitamin C supplementation on acute exercise sessions of low intensity are limited and needed to be explored further.
| Conclusion|| |
In conclusion, our findings suggest that 1000 mg/day of Vitamin C supplementation for 6 weeks decreases BP before and after exercise. The exercise had no effect on any outcomes with and without oral Vitamin C supplementation. The decreased plasma lipid peroxidation biomarker measured using MDA and F2-IsoPs concentrations and increased NO concentration seem to be responsible for the hypotensive effect.
We wish to thank all the participants for their enthusiastic cooperation. We would like to thank Editage (www.editage.com) for English language editing.
Financial support and sponsorship
This study was financially supported by The Royal Golden Jubilee (RGJ) Ph.D. Programme, Thailand Science Research and Innovation. Furthermore, this study was supported by Graduate School Research Grant and Exercise and Sport Sciences Development and Research Group. Blackmores Institute supported products and research expense for this study.
Conflicts of interest
This work was funded to develop a supplement for Blackmores Institute, Australia. However, the company did not involve in the research design, data collection and interpretation, or manuscript writing. No authors received salary from Blackmores Institute.
| References|| |
Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest 2001;108:1341-8.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813-20.
Saxena T, Ali AO, Saxena M. Pathophysiology of essential hypertension: An update. Expert Rev Cardiovasc Ther 2018;16:879-87.
Mamo Y, Bekele F, Nigussie T, Zewudie A. Determinants of poor glycemic control among adult patients with type 2 diabetes mellitus in Jimma University Medical Center, Jimma zone, South West Ethiopia: A case control study. BMC Endocr Disord 2019;19:91.
Asano RY, Sales MM, Browne RA, Moraes JF, Coelho Júnior HJ, Moraes MR, et al.
Acute effects of physical exercise in type 2 diabetes: A review. World J Diabetes 2014;5:659-65.
Asano RY, Sales MM, Coelho JM, Moraes JF, Pereira LA, Campbell CS, et al.
Exercise, nitric oxide, and endothelial dysfunction: A brief review. J Exerc Physiol Online 2012;15:76-86.
Moraes MR, Bacurau RF, Ramalho JD, Reis FC, Casarini DE, Chagas JR, et al.
Increase in kinins on post-exercise hypotension in normotensive and hypertensive volunteers. Biol Chem 2007;388:533-40.
Forjaz CL, Cardoso CG Jr., Rezk CC, Santaella DF, Tinucci T. Postexercise hypotension and hemodynamics: The role of exercise intensity. J Sports Med Phys Fitness 2004;44:54-62.
Simões HG, Asano RY, Sales MM, Browne RA, Arsa G, Motta-Santos D, et al.
Type 2 diabetes elicits lower nitric oxide, bradykinin concentration and kallikrein activity together with higher desArg9-BK and reduced post-exercise hypotension compared to non-diabetic condition. PLoS One 2013;8:e80348.
Lubos E, Handy DE, Loscalzo J. Role of oxidative stress and nitric oxide in atherothrombosis. Front Biosci 2008;13:5323-44.
Giustarini D, Dalle-Donne I, Tsikas D, Rossi R. Oxidative stress and human diseases: Origin, link, measurement, mechanisms, and biomarkers. Crit Rev Clin Lab Sci 2009;46:241-81.
Schwedhelm E, Benndorf RA, Boger RH, Tsikas D. Mass spectrometric analysis of F2-isoprostanes: Markers and mediators in human disease. Curr Pharm Anal 2007;3:39.
Colberg SR, Sigal RJ, Fernhall B, Regensteiner JG, Blissmer BJ, Rubin RR, et al.
Exercise and type 2 diabetes: The American College of sports medicine and the American Diabetes Association: Joint position statement executive summary. Diabetes Care 2010;33:2692-6.
Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage produced by exercise. Biochem Biophys Res Commun 1982;107:1198-205.
Fang ZY, Sharman J, Prins JB, Marwick TH. Determinants of exercise capacity in patients with type 2 diabetes. Diabetes Care 2005;28:1643-8.
Andersen H, Nielsen S, Mogensen CE, Jakobsen J. Muscle strength in type 2 diabetes. Diabetes 2004;53:1543-8.
Wackers FJ, Young LH, Inzucchi SE, Chyun DA, Davey JA, Barrett EJ, et al
. Detection of silent myocardial ischemia in asymptomatic diabetic subjects: The DIAD study. Diabetes Care 2004;27:1954-61.
Tamari Y, Nawata H, Inoue E, Yoshimura A, Yoshii H, Kashino G, et al.
Protective roles of ascorbic acid in oxidative stress induced by depletion of superoxide dismutase in vertebrate cells. Free Radic Res 2013;47:1-7.
Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A 1989;86:6377-81.
Judprasong K, Puwastien P, Rojroongwasinkul N, Nitithamyong A, Sridonpai P, Somjai A. Institute of Nutrition, Mahidol University (2015). Thai Food Composition Database, Online version 2, September 2018, Thailand. http://www.inmu.mahidol.ac.th/thaifcd
Abramson JH. WINPEPI updated: Computer programs for epidemiologists, and their teaching potential. Epidemiol Perspect Innov 2011;8:1.
Mullan BA, Young IS, Fee H, McCance DR. Ascorbic acid reduces blood pressure and arterial stiffness in type 2 diabetes. Hypertension 2002;40:804-9.
Boonthongkaew C, Tong-Un T, Kanpetta Y, Chaungchot N, Macdonald I, Leelayuwat C, et al
. Effects of oral vitamin C treatment on metabolism at rest and in response to an acute exercise in patients with poorly controlled type 2 diabetes mellitus. Arch Allied Health Sci 2020;32:36-50.
Vischer AS, Burkard T. Principles of blood pressure measurement-current techniques, office versus ambulatory blood pressure measurement. Adv Exp Med Biol 2017;956:85-96.
Zhang H, Li J, Wang K, Du X, Li Q. A simple and sensitive assay for ascorbate using potassium ferricyanide as spectroscopic probe reagent. Anal Biochem 2009;388:40-6.
Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M, et al.
A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med 1993;15:353-63.
Cook BG, Cook L, Therrien WJ. Group-difference effect sizes: Gauging the practical importance of findings from group-experimental research. Learn Disabil Res Pract 2018;33:56-63.
Kurl S, Tuomainen TP, Laukkanen JA, Nyyssönen K, Lakka T, Sivenius J, et al.
Plasma vitamin C modifies the association between hypertension and risk of stroke. Stroke 2002;33:1568-73.
Block G, Jensen CD, Norkus EP, Hudes M, Crawford PB. Vitamin C in plasma is inversely related to blood pressure and change in blood pressure during the previous year in young black and white women. Nutr J 2008;7:35.
Lima LC, Assis GV, Hiyane W, Almeida WS, Arsa G, Baldissera V, et al.
Hypotensive effects of exercise performed around anaerobic threshold in type 2 diabetic patients. Diabetes Res Clin Pract 2008;81:216-22.
Tessier DM, Khalil A, Trottier L, Fülöp T. Effects of vitamin C supplementation on antioxidants and lipid peroxidation markers in elderly subjects with type 2 diabetes. Arch Gerontol Geriatr 2009;48:67-72.
Mason SA, Rasmussen B, van Loon LJ, Salmon J, Wadley GD. Ascorbic acid supplementation improves postprandial glycaemic control and blood pressure in individuals with type 2 diabetes: Findings of a randomized cross-over trial. Diabetes Obes Metab 2019;21:674-82.
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, et al.
Vitamin C as an antioxidant: Evaluation of its role in disease prevention. J Am Coll Nutr 2003;22:18-35.
Balbi ME, Tonin FS, Mendes AM, Borba HH, Wiens A, Fernandez-Llimos F, et al.
Antioxidant effects of vitamins in type 2 diabetes: A meta-analysis of randomized controlled trials. Diabetol Metab Syndr 2018;10:18.
Mahmoudabadi MM, Rahbar AR. Effect of EPA and vitamin C on superoxide dismutase, glutathione peroxidase, total antioxidant capacity and malondialdehyde in type 2 diabetic patients. Oman Med J 2014;29:39-45.
Chen H, Karne RJ, Hall G, Campia U, Panza JA, Cannon RO 3rd
, et al.
High-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance. Am J Physiol Heart Circ Physiol 2006;290:H137-45.
Lu Q, Björkhem I, Wretlind B, Diczfalusy U, Henriksson P, Freyschuss A, et al.
Effect of ascorbic acid on microcirculation in patients with type II diabetes: A randomized placebo-controlled cross-over study. Clin Sci (Lond) 2005;108:507-13.
Darko D, Dornhorst A, Kelly FJ, Ritter JM, Chowienczyk PJ. Lack of effect of oral vitamin C on blood pressure, oxidative stress and endothelial function in type II diabetes. Clin Sci (Lond) 2002;103:339-44.
Zhang XM, Ellis EF. Superoxide dismutase decreases mortality, blood pressure, and cerebral blood flow responses induced by acute hypertension in rats. Stroke 1991;22:489-94.
Kontos HA, Wei EP, Dietrich WD, Navari RM, Povlishock JT, Ghatak NR, et al
. Mechanism of cerebral arteriolar abnormalities after acute hypertension. Am J Physiol 1981;240:H511-27.
Nakazono K, Watanabe N, Matsuno K, Sasaki J, Sato T, Inoue M. Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci U S A 1991;88:10045-8.
Huang A, Vita JA, Venema RC, Keaney JF Jr. Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 2000;275:17399-406.
d'Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 2003;92:88-95.
Visioli F, Smith A, Zhang W, Keaney JF Jr., Hagen T, Frei B, et al.
Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status. Redox Rep 2002;7:223-7.
Kaneko T, Kaji K, Matsuo M. Protective effect of lipophilic derivatives of ascorbic acid on lipid peroxide-induced endothelial injury. Arch Biochem Biophys 1993;304:176-80.
Ronchetti SA, Machiavelli LI, Quinteros FA, Duvilanski BH, Cabilla JP. Nitric oxide plays a key role in ovariectomy-induced apoptosis in anterior pituitary: Interplay between nitric oxide pathway and estrogen. PLoS One 2016;11:e0162455.
Coburn SB, Stanczyk FZ, Falk RT, McGlynn KA, Brinton LA, Sampson J, et al.
Comparability of serum, plasma, and urinary estrogen and estrogen metabolite measurements by sex and menopausal status. Cancer Causes Control 2019;30:75-86.
Hellman L, Burns JJ. Metabolism of L-ascorbic acid-1-C14
in man. J Biol Chem 1958;230:923-30.
Kallner A, Horing D, Hartman D. Kinetics of ascorbic acid in humans. In: Seib PA, Tolbert BM, editor Ascorbic acid: Chemistry, metabolism and uses. Advances in Chemistry Series No. 200 American Chemical Society Washington DC 1982: pp 385–400.
[Figure 1], [Figure 2], [Figure 3]
|This article has been cited by|
||Vascular benefits of vitamin C supplementation against fine particulate air pollution in healthy adults: A double-blind randomised crossover trial
| ||Jingyi Ren, Jufeng Liang, Jiaqi Wang, Bowen Yin, Fan Zhang, Xiang Li, Siqi Zhu, Hao Tian, Qiqi Cui, Jianshi Song, Gang Liu, Wenhua Ling, Yuxia Ma |
| ||Ecotoxicology and Environmental Safety. 2022; 241: 113735 |
|[Pubmed] | [DOI]|
||Oxidative stress in the pathophysiology of type 2 diabetes and related complications: Current therapeutics strategies and future perspectives
| ||Jasvinder Singh Bhatti, Abhishek Sehrawat, Jayapriya Mishra, Inderpal Singh Sidhu, Umashanker Navik, Naina Khullar, Shashank Kumar, Gurjit Kaur Bhatti, P. Hemachandra Reddy |
| ||Free Radical Biology and Medicine. 2022; |
|[Pubmed] | [DOI]|