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Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids
Clinical Nutrition xxx (2015) 1e12
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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Meta-analyses
Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids Ammar W. Ashor a, b, *, Mario Siervo a, c, Femke van der Velde a, Naomi D. Willis a, John C. Mathers a a b c
Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle on Tyne NE4 5PL, UK College of Medicine, University of Al-Mustansiriyah, Baghdad, Iraq MRC Human Nutrition Research, Elsie Widdowson Laboratories, 120 Fulbourn Road, Cambridge CB1 9NL, UK
a r t i c l e i n f o
s u m m a r y
Article history: Received 13 January 2015 Accepted 30 May 2015
Background & aims: Randomised controlled trials (RCTs) in humans revealed contradictory results regarding the effect of vitamin C supplementation on blood lipids. We aimed to conduct a systematic review and meta-analysis of RCTs investigating the effect of vitamin C supplementation on total cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglycerides and to determine whether the effects are modified by the participants' or intervention characteristics. Methods: Four databases (PubMed, Embase, Scopus and Cochrane Library) were searched from inception until August 2014 for RCTs supplementing adult participants with vitamin C for 2 weeks and reporting changes in blood lipids. Results: Overall, vitamin C supplementation did not change blood lipids concentration significantly. However, supplementation reduced total cholesterol in younger participants (52 years age) (0.26 mmol/L, 95% CI: 0.45, 0.07) and LDL-C in healthy participants (0.32 mmol/L, 95% CI: 0.57, 0.07). In diabetics, vitamin C supplementation reduced triglycerides significantly (0.15 mmol/L, 95% CI: 0.30, 0.002) and increased HDL-C significantly (0.06 mmol/L, 95% CI: 0.02, 0.11). Meta-regression analyses showed the changes in total cholesterol (b: 0.24, CI: 0.36, 0.11) and in triglycerides (b: 0.17, CI: 0.30, 0.05) following vitamin C supplementation were greater in those with higher concentrations of these lipids at baseline. Greater increase in HDL-C was observed in participants with lower baseline plasma concentrations of vitamin C (b: 0.002, CI: 0.003, 0.0001). Conclusions: Overall, vitamin C supplementation had no significant effect on lipid profile. However, subgroup and sensitivity analyses showed significant reductions in blood lipids following supplementation in sub-populations with dyslipidaemia or low vitamin C status at baseline. PROSPERO Database registration: CRD42014013487, http://www.crd.york.ac.uk/prospero/. © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Ascorbic acid Cholesterol Triglycerides Lipoproteins Nutritional supplements Cardiovascular risk
1. Introduction Modifiable risk factors, notably high blood pressure, dyslipidaemia and hyperglycaemia are major contributors to cardiovascular morbidity and mortality. Importantly, manipulation of these modifiable factors changes the rate of occurrence of cardiovascular
* Corresponding author. Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle on Tyne NE4 5PL, UK. Tel.: þ44 0191 248 1131. E-mail address: [email protected] (A.W. Ashor).
diseases (CVDs) [1]. A meta-analysis of randomised controlled trials (RCTs) suggested that a 40% reduction in low-density lipoprotein cholesterol (LDL-C) with a 30% increase in high-density lipoprotein cholesterol (HDL-C) could lower cardiovascular risk by 70% [2]. Since total cholesterol, LDL-C, HDL-C and, to a lesser extent, triglycerides concentrations are strongly predictive of cardiovascular mortality and morbidity [3], these lipids are useful surrogate outcomes when investigating potential CVD prevention agents. Observational studies indicate that higher vitamin C (ascorbic acid) concentrations are associated with reduced CVD risk [4,5]. Vitamin C is a cofactor for several enzyme-catalysed reactions including the hydroxylation of proline and lysine, essential for the
http://dx.doi.org/10.1016/j.clnu.2015.05.021 0261-5614/© 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
Fig. 1. Overview of possible mechanisms through which vitamin C supplementation may reduce serum lipids concentration.
synthesis of collagen [6], and has roles in radical scavenger activity and nitric oxide-sparing function [7]. Vitamin C is a cofactor for the enzyme 7-a-hydroxylase which is the rate-limiting enzyme in bile acid synthesis [8]. In the liver,
conversion of cholesterol to bile acids may enhance the expression of LDL receptors on hepatocytes (Fig. 1). This is expected to lead to increased LDL-C uptake from the circulation and, eventually, to lower concentrations of LDL-C in blood [9]. Since vitamin C
Fig. 2. Selection of studies for the systematic review and meta-analysis.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
Author
Study design
Abdollahzad et al., 2009 [52] Aro et al., 1988 200 mg [23] Aro et al., 1988 2000 mg [23] Askari et al., 2013 [25] Bhatt et al., 2012 [34] Bishop et al., 1984 [38] Bo et al., 2007 [26] Bordia et al., 1980 [43] Bostom et al., 1995 [12] Cerna et al., 1992 [27] Chaudhari et al., 2012 [35] Dobson et al., 1984 [11] Fotherby et al., 2000 [28] Fuller et al., 2000 [46] Gatto et al., 1996 [29] Ginter et al., 1978 [39] Gokce et al., 1999 [44] Gutierrez et al., 2013 [40] Ingole et al., 2011 [57] Jacques et al., 1995 [30] Khajehdehi et al., 2000 [53] Kim et al., 2004 [31] Lu et al., 2005 [41]
P, DB
Magen et al., 2004 [50] Mahmoudabadi et al., 2011 [42] Mazloom et al., 2011 [36] Menne et al., 1975 [18] Mostafa et al., 1989 [19] Mulholland et al., 1996 [47] Nyyssonen et al., 1997 [24] Nyyssonen et al., 1997 SR [24] Osilesi et al., 1991 [55] Paolisso et al., 1995 [13] Ramos et al., 2008 [54]
Sample size
Health status
Age (years)
BMI (kg/m2)
42
CKD
60
21.6
C, DB
27
CVA
C, DB
27
P, DB
Male %
Vitamin C dose (mg/day)
Duration of intervention (weeks)
Baseline plasma concentration of vitamin C (mm/L)
Baseline TC (mmol/L)
Baseline TG (mmol/L)
Baseline LDL-C (mmol/L)
Baseline HDL-C (mmol/L)
Total score
1.83
1.20
3
52
125
12
14.2
3.62
1.31
81
0
200
6
23.8
5.36
1.28
1.16
4
CVA
81
0
2000
6
23.8
5.36
1.28
1.16
4
30
Healthy
21
22.3
100
500
8
1.61
4
P, UB
59
Type 2 diabetes
57.5
25.8
29
500
12
1.34
3
C, DB
50
Type 2 diabetes
60
48
500
8
UB DB DB UB DB
80 30 43 140 70
Healthy CAD CAD Healthy Type 2 diabetes
34 51 54.7 48 48.3
31 100 60 41 41
2000 1500 4500 500 1000
P, UB C, DB P, DB C, DB P, DB P, DB C, DB
19 40 14 10 48 46 8
Healthy Healthy Smokers Healthy Type 2 diabetes CAD Type 2 diabetes
29 72 20 22.1 55 56 49
53 50 29 0 60 91 50
P, UB P, DB P, UB
30 138 39
Schizophrenia Healthy CKD
25.3 41 31.4
19.4 25.8
P, DB C, DB
305 17
56.6 54
P, UB P, DB
33 34
Healthy Type 2 diabetes Hyperlipidaemia Type 2 diabetes
SB UB DB UB
27 122 67 14
P, SB
5.09
1.81
49.4
6.8
2.35
2 24 12 72 12
53.4
0.8 1.75
56.8 56.0 14.8
4.9 7.25 5.84 7.28 6.18
1000 500 1000 1000 500 500 1000
24 12 8 8 48 4 2
55.0 49.0 39.0 62.0 21.6 41.0 45.4
5.6 6.2 4.4 5.1 7.98 5.36 4.20
1.6 1.11 2.11 2.40 2.11
37 44 51
1000 1000 200
6 32 12
72.0
4.21 4.87 6.23
23.2
36 71
500 3000
240 2
64.0 26.3
52.6 52.6
28.2 29.3
52 100
500 200
8 8
Type 2 diabetes Healthy Healthy Smokers
47 21.5
26.9
30 89 60 0
1000 1000 500 1000
6 16 24 2
29
Smokers
50
100
500
8
P, SB
30
Smokers
50
100
500
C, DB
20
Pre-hypertensive
57.8
30.2
25
C, DB
40
Type 2 diabetes
72
27.6
P, UB
34
CKD
24.5
P, P, P, P, P,
P, P, P, P,
21.7 30
21.6 29.8 29.4
2.50
3 1.4
3 4 4 2 5
4.76 4.27
0.97 1.42 0.98
3.6 2.4 2.8
1.53 1.3 1.76
3.44 2.36
1.04 0.96
2 4 3 2 3 4 4
1.36 1.17 5.66
2.43 3.08 4.4
1.15 1.19 0.92
2 3 2
5.3 5.2
1.29 2.2
3.18
1.55 1.1
3 4
4.47
1.85
4.07 2.70
0.99
2 3
4.91 4.49 5.07
1.98 1.31 1.10
3.50
0.81
3.41 4.6
1.14
63.0
1.57
4.2
1.18
3
8
61.0
1.87
4.13
1.01
3
1000
6
74.0
5.04
1.45
1.15
3
48
1000
16
41.0
7.2
2.61
5.7
1.1
3
68
1000
48
4.75
1.50
2.60
1.30
2
73.8
2.58 2.15
37.0
A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
Table 1 Characteristics of the studies included in the systematic review and meta-analysis.
5 3 3 2
(continued on next page) 3
21
63
26
18
34
18
8
P, DB
P, SB
C, DB
C, DB
C, DB
P, UB
Healthy
36 P, DB
Healthy
16 P, DB
Schindler et al., 2003 [56] Shidfar et al., 2003 [51] Singhal et al., 2001 [45] Tousoulis et al., 2007 [37] Tofler et al., 2000 [32] Van Hoydonck et al., 2004 [48] Vinson and Jang 2001 [49] Yanai and Morimoto 2004 [33]
BMI, body mass index; CAD, coronary artery diseases; CVA, cerebrovascular accident; CKD, chronic kidney diseases; C, crossover; DB, double-blind; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; P, parallel; SB, single-blind; TC, total cholesterol; TG, triglycerides; UB, unblinded.
2 2.4 3 100 21
1000
8 56 53 Hyperlipidaemia
1000
4 500 100 52 Smokers
25.1
100 40
2000
6
1.1
5 7.23
2.3
3
3.50 5.52
1.34
3 47.0
1.35
3 78.9
3 1.36 1.69 29.1 59.1 Type 2 diabetes
54
2000
4
4.88
2.64
3 1.13 2.42 24.3 54.7 CAD
71
1000
4
5.37
3.13
3 0.96 3.56 6.31 27.8 51.8 Hyperlipidaemia
36
500
10
74.9
4.16
1.27 24.7 63 Hypertensive
63
2000
96
1.51 5.15 54.5
3.44
Baseline HDL-C (mmol/L) Baseline LDL-C (mmol/L) Baseline TG (mmol/L) Baseline TC (mmol/L) Baseline plasma concentration of vitamin C (mm/L) Duration of intervention (weeks) Vitamin C dose (mg/day) Male % BMI (kg/m2) Age (years) Health status Sample size Study design Author
Table 1 (continued )
3
A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
Total score
4
facilitates the conversion of cholesterol into bile acids it may help lower blood cholesterol concentrations [10]. Another possible mechanism involves the action of vitamin C as an antioxidant. By inhibiting the oxidation of LDL-C, vitamin C facilitates its binding to, and uptake by, LDL receptors on hepatocytes (Fig. 1) [8]. Animals deficient in vitamin C exhibit hypercholesterolaemia and the subsequent administration of vitamin C may restore cholesterol concentrations to normal [10]. Furthermore, human observational studies showed that high plasma concentrations of vitamin C are associated with more favourable lipid profiles [5,11]. However, RCTs have produced contradictory results about the effect of vitamin C supplementation on blood lipids [12,13] and there is no evidence from RCTs that supplemental vitamin C reduces CVD risk [14]. A previous meta-analysis of 13 trials which recruited participants with hypercholesterolaemia showed that vitamin C supplementation reduced concentrations of LDL-C and triglycerides significantly with no significant effect on HDL-C concentration [8]. The present study aimed to conduct a systematic review and meta-analysis of RCTs investigating the effect of vitamin C supplementation on blood lipid (total cholesterol, LDL-C, HDL-C and triglycerides) concentrations. The secondary aim of the study was to determine whether the effects of such supplementation were modified by the participants' characteristics (health status, age, body mass index [BMI], baseline concentration of serum lipids and baseline concentration of vitamin C) or by features of the intervention (e.g. dose or duration of vitamin C administration). 2. Methods The present systematic review was conducted according to the Cochrane guidelines [15] and it is reported according to PRISMA guidelines [16]. 2.1. Literature search Four databases (PubMed, Embase, Scopus and Cochrane Library) were used to search for articles from inception until August 2014. In addition, a manual search of the reference lists of relevant reviews and articles included in the systematic review was performed. The following terms and keywords were used: vitamin C, ascorbic acid, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, triacylglycerol and randomised controlled trial. 2.2. Study selection The following criteria were applied to identify articles to be included in this systematic review and meta-analysis: 1) randomised controlled trials (no exclusion criteria were applied in relation to study design or blinding); 2) studies involving adults aged 18 years (no exclusion was applied for health status, smoking history or body size); 3) vitamin C administered alone, i.e. not combined with other drugs or nutritional interventions; 4) duration of intervention 2 weeks; 5) studies reporting changes in lipid profile (concentrations of total cholesterol, LDL-C, HDL-C and triglycerides); 6) English-language restriction but not time restriction was applied. Two investigators (FVV, NDW) independently screened the titles and abstracts of the articles to evaluate eligibility for inclusion. If consensus was reached, articles were either excluded or moved to the next stage (full-text). If consensus was not reached, the article was moved to the full-text stage and was appraised critically to determine eligibility for inclusion in the systematic review. Disagreements were resolved by discussion between the reviewers until consensus was reached.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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2.3. Data extraction and quality assessment
2.4. Statistical analysis
The following information was extracted independently by two investigators (AWA, FVV) from the eligible articles: 1) authors and year of publication; 2) study characteristics (design, sample size); 3) participant characteristics (age, male/female ratio, health status and baseline values for BMI, lipids concentration and plasma concentration of vitamin C) and 4) dose and duration of vitamin C intervention. Any disagreements in data extraction were resolved through discussion. In addition, the modified Jadad scale was adopted to assess the risk of bias of the included studies. Three main items: randomisation, blinding and description of dropouts or withdrawals were used to rate the quality of the included studies [17]. Possible scores ranged from 0 to 5 and a score of <3 indicates high risk while a score of 3 indicates low risk of bias.
The outcome of the meta-analysis is the net difference in lipids concentration (total cholesterol, LDL-C, HDL-C and triglycerides) at the end of the study between the intervention and control groups. Random effect models were used to take into account heterogeneity among studies. Forest plots were generated for graphical presentation of the serum lipid outcomes. Statistical analyses were performed using STATA 12 (StataCorp. 2011, College Station, TX, USA). The effect size was estimated as weighted mean differences (WMDs) with 95% confidence interval by using inverse variance weighting. Data not provided in the main text or tables were extracted from the figures. One study did not report the values for the standard deviations (SDs) [18]; the missing values were imputed according to the method described by Furukawa et al. [6]. Another study did not report blood lipids concentration at the end of
Fig. 3. Forest plot summarising the effect of vitamin C supplementation on total cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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intervention for the placebo group [19]; in this case we substituted the baseline concentrations as the values for blood lipids at the end of intervention. For crossover trials, we used the mean and SD separately for the intervention and control conditions. This is regarded as a conservative approach that will reduce the power of these studies to show the true effect of the intervention [20]. In trials with multiple treatment arms and a single control group, the sample size of the control group was divided by the number of treatment groups to avoid over-inflation of the sample size [15]. Subgroup analyses were performed to identify possible sources of heterogeneity. The factors used in subgroup analyses included participant characteristics such as health status, age, baseline BMI, blood lipids and plasma concentration of vitamin C (median values). Further subgroup analyses were conducted according to study characteristics (dose, duration of vitamin C intervention and design and quality of the included studies). However, in this investigation we restricted subgroup analysis to cases with 5 studies per subgroup [15]. Meta-regression analysis was used to determine whether age, BMI, vitamin C dose, baseline concentration of serum lipids and of vitamin C influenced changes in the serum lipid profile in response to supplementation. Publication bias was evaluated by visual inspection of the funnel plot and by Egger's regression test [21]. Heterogeneity between studies was evaluated using Cochrane Q statistics; P > 0.1 indicates significant heterogeneity. The I2 test was also used to evaluate consistency between studies where a value <25% indicates low risk of heterogeneity, 25e75% indicates moderate risk of heterogeneity and >75% indicates high risk of heterogeneity [22].
3. Results 3.1. Search results The process of screening and selection of the studies is summarised in Fig. 2. The primary search of the four databases produced 1013 articles after the removal of duplicates. Additionally, three studies were found by manually searching the references of relevant reviews and studies. After title and abstract screening, 144 full-text papers were retrieved for further evaluation and a total of 40 publications were identified which met the inclusion criteria for entry into the systematic review. 3.2. Characteristics of the studies The total number of participants from 40 studies included in this systematic review was 1981 (1049 males, 932 females) with a median of 33 (range 8e305) participants per study. The overall median age was 52 (range 20e81) years. Trial designs included 29 parallel groups and 11 crossover studies. Two studies had two independent subgroups [23,24], therefore the total number of trials included was 42 (Table 1). Thirty-seven trials reported the effect of vitamin C supplementation on serum cholesterol, 35 on serum triglycerides, 29 on serum LDL-C and 35 on serum HDL-C concentrations. The health status of the participants in the trials differed; 12 trials included healthy individuals [11,18,19,25e33], 10 trials recruited diabetics [13,34e42], four recruited those with coronary artery disease [12,43e45], four trials recruited smokers [24,46e48],
Table 2 Pooled estimates of the effect of vitamin C supplementation on total cholesterol, LDL-C, HDL-C and triglycerides concentrations within various subgroups.
Age of participants (years)a 52 >52 Body mass index (kg/m2)a 26 >26 Baseline serum lipids (mmol/L)a TC 5.3; TG 1.7; LDL 3.4; HDL 1.16 TC > 5.3; TG > 1.7; LDL > 3.4; HDL > 1.16 Vitamin C dose (mg/day)a 1000 >1000 Plasma vitamin C concentration (mm/L) at baselinea 49.4 >49.4 Intervention duration (weeks)a 8 >8 Study Design Parallel Crossover Study quality (Jadad scale) Low risk > 3 High risk 3
Total cholesterol
LDL cholesterol
HDL cholesterol
Triglycerides
No. groups
No. groups
No. groups
No. groups
Effect size (95% CI) (P value) P ¼ 0.03
Effect size (95% CI) (P value) P ¼ 0.60
Effect size (95% CI) (P value) P ¼ 0.84
Effect size (95% CI) (P value) P ¼ 0.61
16 18
0.26 (0.45, 0.07) 0.03 (0.18, 0.13) P ¼ 0.58
15 12
0.13 (0.40, 0.13) 0.06 (0.39, 0.28) P ¼ 0.29
17 16
0.005 (0.05, 0.04) 0.001 (0.05, 0.05) P ¼ 0.27
18 15
0.04 (0.11, 0.03) 0.01 (0.23, 0.21) P ¼ 0.32
11 9
0.02 (0.12, 0.16) 0.13 (0.50, 0.23) P ¼ 0.06
11 7
0.13 (0.43, 0.17) 0.26 (0.41, 0.94) P ¼ 0.95
13 8
0.01 (0.05, 0.07) 0.07 (0.16, 0.01) P ¼ 0.78
12 8
0.04 (0.13, 0.20) 0.12 (0.37, 0.13) P ¼ 0.11
18
0.02 (0.12, 0.14)
16
0.06 (0.30, 0.19)
17
0.001 (0.04, 0.04)
17
0.05 (0.07, 0.18)
17
0.30 (0.51, 0.09)
12
0.10 (0.46, 0.26)
17
0.02 (0.04, 0.08)
16
0.14 (0.28, 0.002)
29 8
P ¼ 0.19 0.13 (0.26, 0.002) 0.01 (0.27, 0.25) P ¼ 0.46
26 3
P ¼ 0.07 0.15 (0.32, 0.02) 0.46 (0.24, 1.15) P ¼ 0.11
28 7
P ¼ 0.42 0.01 (0.02, 0.05) 0.03 (0.13, 0.07) P ¼ 0.06
28 7
P ¼ 0.26 0.06 (0.15, 0.03) 0.05 (0.26, 0.35) P ¼ 0.10
14 12
0.19 (0.30, 0.09) 0.17 (0.39, 0.04) P ¼ 0.61
19 18
0.08 (0.17, 0.20 (0.40, P ¼ 0.24 0.08 (0.23, 0.19 (0.29, P ¼ 0.10 0.06 (0.17, 0.36 (0.85,
25 12 30 7
8 9
0.34 (0.58, 0.09) 0.11 (0.31, 0.53) P ¼ 0.81
11 12
0.05 (0.02, 0.09) 0.03 (0.07, 0.01) P ¼ 0.82
12 12
0.14 (0.28, 0.002) 0.05 (0.12, 0.23) P ¼ 0.57
0.02) 0.01)
16 13
21 14
22 7
0.05) 0.13)
22 7
0.001 (0.05, 0.04) 0.01 (0.04, 0.06) P ¼ 0.57 0.00 (0.04, 0.04) 0.04 (0.004, 0.09) P ¼ 0.96 0.008 (0.02, 0.04) 0.03 (0.09, 0.15)
20 15
0.07) 0.08)
0.04 (0.23, 0.16) 0.15 (0.48, 0.18) P ¼ 0.27 0.03 (0.26, 0.20) 0.09 (0.28, 0.11) P ¼ 0.08 0.01 (0.21, 0.24) 0.41 (0.80, 0.02)
0.03 (0.13, 0.08) 0.08 (0.27, 0.12) P ¼ 0.06 0.03 (0.09, 0.14) 0.17 (0.32, 0.01) P ¼ 0.35 0.005 (0.12, 0.11) 0.12 (0.34, 0.10)
24 11 29 6
25 10 29 6
HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Values sub-grouped by the median; P value for the differences between subgroups.
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three trials recruited patients with hyperlipidaemia [49e51], three trials recruited patients with chronic kidney disease [52e54] and there was one trial for each of the following: cerebrovascular disease [23], pre-hypertensive [55], hypertensive [56] and schizophrenic [57] participants. The duration of vitamin C supplementation ranged from two weeks to five years (median duration: eight weeks) and the dose of vitamin C ranged from 125 to 4500 mg per day (median dose: 1000 mg/day). The quality of the included studies ranged from 2 to 5 (median: 3) on the Jadad scale. On this scoring system, nine studies may have a high risk of bias [11,27,29,33,47,50,53,54,57] whereas the other 31 studies showed a score 3 on the Jadad scale (Table 1). Four studies described the method of randomization [12,26,34,35] and two studies stated the methods of allocation concealment [26,35]. For the crossover trials, most reported the washout period used (range 2e4 weeks) while three studies did not report this information [29,38,40]. Only one study reported the use of lipid-lowering agents during the intervention period by some of the included participants [12].
significantly greater effects of vitamin C supplementation in lowering total cholesterol concentration in those with higher baseline plasma cholesterol concentrations (b: 0.24, CI: 0.36, 0.11, P < 0.01) (Fig. 4A).
4. Meta-analysis
4.3. Effect of vitamin C on HDL-C concentration
4.1. Effect of vitamin C on total cholesterol concentration
Across all studies, vitamin C supplementation had no significant effect on HDL-C concentrations (0.004 mmol/L, 95% CI: 0.03, 0.03, P ¼ 0.83) and there was significant heterogeneity between studies (I2 ¼ 45; P < 0.01) (Fig. 6). However, supplementation improved HDL-C concentration in diabetics (0.06 mmol/L, 95% CI: 0.02, 0.11, P ¼ 0.01) (Supplemental Fig. 2 and Supplemental Table 3, “Supplementary data” in the online issue). Meta-regression analysis showed the change in HDL-C concentrations following vitamin C supplementation was greater in those with low baseline plasma concentrations of vitamin C (b: 0.002, CI: 0.003, 0.0001, P ¼ 0.04) (Fig. 4C).
Overall, vitamin C supplementation was ineffective in reducing total cholesterol concentration (0.10 mmol/L, 95% CI: 0.22, 0.01, P ¼ 0.08) and there was significant heterogeneity between studies (I2 ¼ 57.2; P < 0.01) (Fig. 3). However, subgroup analyses demonstrated a significant reduction in total cholesterol (0.26 mmol/L, 95% CI: 0.45, 0.07, P < 0.01) in younger participants (52 years age) following vitamin C supplementation (Table 2). Further information can be found in Supplemental Table 1 under “Supplementary data” in the online issue. Meta-regression analysis showed
4.2. Effect of vitamin C on LDL-C concentration Overall, there was no significant effect of vitamin C supplementation on serum LDL-C (0.09 mmol/L, 95% CI: 0.28, 0.11, P ¼ 0.39) and there was significant heterogeneity between studies (I2 ¼ 85; P < 0.01) (Fig. 5). However, subgroup analysis showed that vitamin C supplementation reduced LDL-C concentrations significantly in healthy participants (0.32 mmol/L, 95% CI: 0.57, 0.07, P ¼ 0.01) (Supplemental Fig. 1 and Supplemental Table 2, “Supplementary data” in the online issue). There was a trend towards a larger reduction in LDL-C concentration following vitamin C supplementation in participants with low baseline plasma concentrations of vitamin C (b: 0.01, CI: 0.004, 0.03, P ¼ 0.08) (Fig. 4B).
Fig. 4. Associations between baseline concentrations of serum lipids and of vitamin C and the effect of vitamin C supplementation on change in (A) total cholesterol, (B) low-density lipoprotein cholesterol (LDL-C), (C) high-density lipoprotein cholesterol (HDL-C) and (D) triglycerides concentrations. The size of each circle is proportional to the number of participants in that study.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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4.4. Effect of vitamin C on triglycerides concentration
5. Discussion
Overall, there was no significant effect of vitamin C supplementation on serum triglycerides concentration (0.03 mmol/L, 95% CI: 0.13, 0.08, P ¼ 0.62) and there was significant heterogeneity between studies (I2 ¼ 72.4; P < 0.01) (Fig. 7). However, in diabetics, vitamin C supplementation reduced serum triglycerides concentration significantly (0.15 mmol/L, 95% CI: 0.30, 0.002, P ¼ 0.01) (Supplemental Fig. 2 and Supplemental Table 4, “Supplementary data” in the online issue). In addition, meta-regression analysis demonstrated that vitamin C supplementation lowered triglycerides to a greater extent in those with higher triglycerides concentration at baseline (b: 0.17, CI: 0.30, 0.05, P ¼ 0.03) (Fig. 4D).
Overall, vitamin C supplementation had no significant effect on lipid profile. However, supplementation reduced total cholesterol concentration in younger (52 years) participants and LDL-C concentration in healthy participants. In addition, in diabetics, vitamin C supplementation reduced triglyceride and increased HDL-C concentrations significantly. Vitamin C produced bigger falls in total cholesterol and triglycerides in those participants with higher concentrations of these lipids at baseline and greater increase in HDL-C concentration in participants with low baseline plasma concentration of vitamin C. Despite the overall lack of effect of vitamin C supplementation on blood lipids concentration, this meta-analysis demonstrated that there may be specific groups within the population who would benefit from supplementation with vitamin C, including those with habitually low (baseline) vitamin C status or individuals at higher CVD risk (e.g. those with hypercholesterolaemia or dyslipidaemia). Therefore, research on identification of those with potential to benefit might offer an objective basis for individualising such interventions. For example, a previous study showed that supplementation with vitamin C reduced markers of oxidative stress in smokers with high BMI but no effect was seen in normal BMI smokers [58]. Similarly, Khan et al. found a significant improvement in endothelial function after vitamin C supplementation to healthy participants with habitually
4.5. Publication bias Visual inspection of the funnel plots showed no evidence of publication bias for the effect of vitamin C supplementation on total cholesterol (Supplemental Fig. 3A), LDL-C (Supplemental Fig. 3B), HDL-C (Supplemental Fig. 3C) or triglycerides concentration (Supplemental Fig. 3D, to be found online under “Supplementary data”). Egger's regression test outcomes (b: 0.03, P ¼ 0.43; b: 0.05, P ¼ 0.82; b: 0.26, P ¼ 0.16; b: 0.01, P ¼ 0.85 for total cholesterol, LDL-C, HDL-C and triglycerides respectively) confirmed the likely absence of publication bias for all four lipid fractions.
Fig. 5. Forest plot summarising the effect of vitamin C supplementation on low-density lipoprotein cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
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Fig. 6. Forest plot summarising the effect of vitamin C supplementation on high-density lipoprotein cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
low intakes of fruit and vegetables [59]. Furthermore, supplementation with multivitamins for six years in a population with high prevalence of micronutrient deficiency improved cerebrovascular disease mortality significantly [60]. These studies support the concept that individual vitamin status may determine the magnitude of the effect of antioxidant vitamin supplementation on CVD risk factors. In humans, a significant inverse association was found between plasma levels of vitamin C and risk of diabetes (OR ¼ 0.38, 95% CI: 0.28, 0.52) [61]. A recently conducted RCT demonstrated that administration of vitamin C to diabetic individuals resulted in significant reductions in fasting blood glucose and in glycosylated haemoglobin concentrations [62]. The possible mechanisms underlying the association of vitamin C with glucose metabolism include, i) the ability of vitamin C to prevent glycosylation of haemoglobin by inhibiting the formation of advanced glycosylation end products and ii) the ability of vitamin C to mitigate the longterm pancreatic b-cell dysfunction caused by oxidative stress [62]. The beneficial effects on triglyceride and HDL-C concentrations observed in the present meta-analysis might be explained by the ability of vitamin C to counteract the oxidative stress-induced insulin resistance in diabetic individuals. The vitamin C-induced
insulin sensitivity may, therefore, ameliorate the insulin resistanceinduced dyslipidaemia in diabetics (Fig. 1). In the present meta-analysis, a larger reduction in total cholesterol and triglycerides in response to vitamin C supplementation was observed in participants with higher concentrations of these lipids at baseline. This is in accord with a previous metaanalysis which showed significant reductions in LDL-C and triglycerides in individuals with hypercholesterolaemia [8]. Additionally, we observed greater reductions in LDL-C and increases in HDL-C following vitamin C supplementation in participants with lower plasma concentration of vitamin C at baseline. A previous meta-analysis showed that antioxidant vitamin supplementation improved arterial stiffness to a greater extent in individuals with low baseline plasma concentration of vitamin C [63]. Vitamin C supplementation did not alter total cholesterol significantly in older participants (>52 years). Vitamin C absorption may be less efficient in older individuals [64,65] and so higher doses of vitamin C might be required to produce significant beneficial effects on serum lipids. However, the relatively large doses of vitamin C (median 1000 mg/day) used in the studies included in this review make it unlikely that inadequate tissue availability explains the lack of effect of supplementation in older people.
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Fig. 7. Forest plot summarising the effect of vitamin C supplementation on triglycerides concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
A meta-analysis of 14 RCTs encompassing 90,056 participants showed that a mean reduction of 1 mmol in LDL-C is associated with a 23% reduction in myocardial infarction, a 17% reduction in stroke and a 12% reduction in all-cause mortality [66]. Using the outcomes from the present meta-analysis, this can be translated into possible reductions of 2.2%, 1.6% and 1.2% in myocardial infarction, stroke and all-cause mortality, respectively, with vitamin C supplementation. One of the major limitations of the present meta-analysis is the relatively small sample size of the included studies. However, most of the included studies used robust study designs and were rated as moderate/high quality on the Jadad scale. Another limitation is the small number of studies of participants with relevant medical conditions including coronary artery disease, hypertension and chronic kidney disease which limit our ability to draw conclusions regarding the effects of vitamin C supplementation in these conditions. Lastly, considerable heterogeneity was observed between studies which might be due to differences in study design and in the population groups included in these studies. Overall, this systematic review and meta-analysis found that vitamin C supplementation had no significant effect on lipid profile. This finding is in accord with the overall lack of effect of supplemental vitamin C on CVD outcomes observed in large-scale RCTs.
However, subgroup and sensitivity analyses showed that vitamin C supplementation reduced blood lipids significantly in participants with dyslipidaemia or low baseline vitamin C status. The results emphasise the potential importance of a personalised approach to vitamin C interventions in the primary and secondary prevention of CVDs. Future studies are required to confirm the observation that supplemental vitamin C appears to improve dyslipidaemia in individuals with type 2 diabetes and to explore the underlying mechanisms. Conflict of interest None. Acknowledgements AWA is funded by the Ministry of Higher Education and Scientific Research of Iraq. JCM acknowledges support from the LiveWell Programme a research project funded through a collaborative grant from the Lifelong Health and Wellbeing (LLHW) initiative, managed by the Medical Research Council (MRC) (grant number G0900686). The authors have no conflicts of interest to declare. The authors' responsibilities were as follows - AWA: drafted the manuscript;
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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Meta-analyses
Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids Ammar W. Ashor a, b, *, Mario Siervo a, c, Femke van der Velde a, Naomi D. Willis a, John C. Mathers a a b c
Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle on Tyne NE4 5PL, UK College of Medicine, University of Al-Mustansiriyah, Baghdad, Iraq MRC Human Nutrition Research, Elsie Widdowson Laboratories, 120 Fulbourn Road, Cambridge CB1 9NL, UK
a r t i c l e i n f o
s u m m a r y
Article history: Received 13 January 2015 Accepted 30 May 2015
Background & aims: Randomised controlled trials (RCTs) in humans revealed contradictory results regarding the effect of vitamin C supplementation on blood lipids. We aimed to conduct a systematic review and meta-analysis of RCTs investigating the effect of vitamin C supplementation on total cholesterol, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C) and triglycerides and to determine whether the effects are modified by the participants' or intervention characteristics. Methods: Four databases (PubMed, Embase, Scopus and Cochrane Library) were searched from inception until August 2014 for RCTs supplementing adult participants with vitamin C for 2 weeks and reporting changes in blood lipids. Results: Overall, vitamin C supplementation did not change blood lipids concentration significantly. However, supplementation reduced total cholesterol in younger participants (52 years age) (0.26 mmol/L, 95% CI: 0.45, 0.07) and LDL-C in healthy participants (0.32 mmol/L, 95% CI: 0.57, 0.07). In diabetics, vitamin C supplementation reduced triglycerides significantly (0.15 mmol/L, 95% CI: 0.30, 0.002) and increased HDL-C significantly (0.06 mmol/L, 95% CI: 0.02, 0.11). Meta-regression analyses showed the changes in total cholesterol (b: 0.24, CI: 0.36, 0.11) and in triglycerides (b: 0.17, CI: 0.30, 0.05) following vitamin C supplementation were greater in those with higher concentrations of these lipids at baseline. Greater increase in HDL-C was observed in participants with lower baseline plasma concentrations of vitamin C (b: 0.002, CI: 0.003, 0.0001). Conclusions: Overall, vitamin C supplementation had no significant effect on lipid profile. However, subgroup and sensitivity analyses showed significant reductions in blood lipids following supplementation in sub-populations with dyslipidaemia or low vitamin C status at baseline. PROSPERO Database registration: CRD42014013487, http://www.crd.york.ac.uk/prospero/. © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Ascorbic acid Cholesterol Triglycerides Lipoproteins Nutritional supplements Cardiovascular risk
1. Introduction Modifiable risk factors, notably high blood pressure, dyslipidaemia and hyperglycaemia are major contributors to cardiovascular morbidity and mortality. Importantly, manipulation of these modifiable factors changes the rate of occurrence of cardiovascular
* Corresponding author. Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle on Tyne NE4 5PL, UK. Tel.: þ44 0191 248 1131. E-mail address: [email protected] (A.W. Ashor).
diseases (CVDs) [1]. A meta-analysis of randomised controlled trials (RCTs) suggested that a 40% reduction in low-density lipoprotein cholesterol (LDL-C) with a 30% increase in high-density lipoprotein cholesterol (HDL-C) could lower cardiovascular risk by 70% [2]. Since total cholesterol, LDL-C, HDL-C and, to a lesser extent, triglycerides concentrations are strongly predictive of cardiovascular mortality and morbidity [3], these lipids are useful surrogate outcomes when investigating potential CVD prevention agents. Observational studies indicate that higher vitamin C (ascorbic acid) concentrations are associated with reduced CVD risk [4,5]. Vitamin C is a cofactor for several enzyme-catalysed reactions including the hydroxylation of proline and lysine, essential for the
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Fig. 1. Overview of possible mechanisms through which vitamin C supplementation may reduce serum lipids concentration.
synthesis of collagen [6], and has roles in radical scavenger activity and nitric oxide-sparing function [7]. Vitamin C is a cofactor for the enzyme 7-a-hydroxylase which is the rate-limiting enzyme in bile acid synthesis [8]. In the liver,
conversion of cholesterol to bile acids may enhance the expression of LDL receptors on hepatocytes (Fig. 1). This is expected to lead to increased LDL-C uptake from the circulation and, eventually, to lower concentrations of LDL-C in blood [9]. Since vitamin C
Fig. 2. Selection of studies for the systematic review and meta-analysis.
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Author
Study design
Abdollahzad et al., 2009 [52] Aro et al., 1988 200 mg [23] Aro et al., 1988 2000 mg [23] Askari et al., 2013 [25] Bhatt et al., 2012 [34] Bishop et al., 1984 [38] Bo et al., 2007 [26] Bordia et al., 1980 [43] Bostom et al., 1995 [12] Cerna et al., 1992 [27] Chaudhari et al., 2012 [35] Dobson et al., 1984 [11] Fotherby et al., 2000 [28] Fuller et al., 2000 [46] Gatto et al., 1996 [29] Ginter et al., 1978 [39] Gokce et al., 1999 [44] Gutierrez et al., 2013 [40] Ingole et al., 2011 [57] Jacques et al., 1995 [30] Khajehdehi et al., 2000 [53] Kim et al., 2004 [31] Lu et al., 2005 [41]
P, DB
Magen et al., 2004 [50] Mahmoudabadi et al., 2011 [42] Mazloom et al., 2011 [36] Menne et al., 1975 [18] Mostafa et al., 1989 [19] Mulholland et al., 1996 [47] Nyyssonen et al., 1997 [24] Nyyssonen et al., 1997 SR [24] Osilesi et al., 1991 [55] Paolisso et al., 1995 [13] Ramos et al., 2008 [54]
Sample size
Health status
Age (years)
BMI (kg/m2)
42
CKD
60
21.6
C, DB
27
CVA
C, DB
27
P, DB
Male %
Vitamin C dose (mg/day)
Duration of intervention (weeks)
Baseline plasma concentration of vitamin C (mm/L)
Baseline TC (mmol/L)
Baseline TG (mmol/L)
Baseline LDL-C (mmol/L)
Baseline HDL-C (mmol/L)
Total score
1.83
1.20
3
52
125
12
14.2
3.62
1.31
81
0
200
6
23.8
5.36
1.28
1.16
4
CVA
81
0
2000
6
23.8
5.36
1.28
1.16
4
30
Healthy
21
22.3
100
500
8
1.61
4
P, UB
59
Type 2 diabetes
57.5
25.8
29
500
12
1.34
3
C, DB
50
Type 2 diabetes
60
48
500
8
UB DB DB UB DB
80 30 43 140 70
Healthy CAD CAD Healthy Type 2 diabetes
34 51 54.7 48 48.3
31 100 60 41 41
2000 1500 4500 500 1000
P, UB C, DB P, DB C, DB P, DB P, DB C, DB
19 40 14 10 48 46 8
Healthy Healthy Smokers Healthy Type 2 diabetes CAD Type 2 diabetes
29 72 20 22.1 55 56 49
53 50 29 0 60 91 50
P, UB P, DB P, UB
30 138 39
Schizophrenia Healthy CKD
25.3 41 31.4
19.4 25.8
P, DB C, DB
305 17
56.6 54
P, UB P, DB
33 34
Healthy Type 2 diabetes Hyperlipidaemia Type 2 diabetes
SB UB DB UB
27 122 67 14
P, SB
5.09
1.81
49.4
6.8
2.35
2 24 12 72 12
53.4
0.8 1.75
56.8 56.0 14.8
4.9 7.25 5.84 7.28 6.18
1000 500 1000 1000 500 500 1000
24 12 8 8 48 4 2
55.0 49.0 39.0 62.0 21.6 41.0 45.4
5.6 6.2 4.4 5.1 7.98 5.36 4.20
1.6 1.11 2.11 2.40 2.11
37 44 51
1000 1000 200
6 32 12
72.0
4.21 4.87 6.23
23.2
36 71
500 3000
240 2
64.0 26.3
52.6 52.6
28.2 29.3
52 100
500 200
8 8
Type 2 diabetes Healthy Healthy Smokers
47 21.5
26.9
30 89 60 0
1000 1000 500 1000
6 16 24 2
29
Smokers
50
100
500
8
P, SB
30
Smokers
50
100
500
C, DB
20
Pre-hypertensive
57.8
30.2
25
C, DB
40
Type 2 diabetes
72
27.6
P, UB
34
CKD
24.5
P, P, P, P, P,
P, P, P, P,
21.7 30
21.6 29.8 29.4
2.50
3 1.4
3 4 4 2 5
4.76 4.27
0.97 1.42 0.98
3.6 2.4 2.8
1.53 1.3 1.76
3.44 2.36
1.04 0.96
2 4 3 2 3 4 4
1.36 1.17 5.66
2.43 3.08 4.4
1.15 1.19 0.92
2 3 2
5.3 5.2
1.29 2.2
3.18
1.55 1.1
3 4
4.47
1.85
4.07 2.70
0.99
2 3
4.91 4.49 5.07
1.98 1.31 1.10
3.50
0.81
3.41 4.6
1.14
63.0
1.57
4.2
1.18
3
8
61.0
1.87
4.13
1.01
3
1000
6
74.0
5.04
1.45
1.15
3
48
1000
16
41.0
7.2
2.61
5.7
1.1
3
68
1000
48
4.75
1.50
2.60
1.30
2
73.8
2.58 2.15
37.0
A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
Table 1 Characteristics of the studies included in the systematic review and meta-analysis.
5 3 3 2
(continued on next page) 3
21
63
26
18
34
18
8
P, DB
P, SB
C, DB
C, DB
C, DB
P, UB
Healthy
36 P, DB
Healthy
16 P, DB
Schindler et al., 2003 [56] Shidfar et al., 2003 [51] Singhal et al., 2001 [45] Tousoulis et al., 2007 [37] Tofler et al., 2000 [32] Van Hoydonck et al., 2004 [48] Vinson and Jang 2001 [49] Yanai and Morimoto 2004 [33]
BMI, body mass index; CAD, coronary artery diseases; CVA, cerebrovascular accident; CKD, chronic kidney diseases; C, crossover; DB, double-blind; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; P, parallel; SB, single-blind; TC, total cholesterol; TG, triglycerides; UB, unblinded.
2 2.4 3 100 21
1000
8 56 53 Hyperlipidaemia
1000
4 500 100 52 Smokers
25.1
100 40
2000
6
1.1
5 7.23
2.3
3
3.50 5.52
1.34
3 47.0
1.35
3 78.9
3 1.36 1.69 29.1 59.1 Type 2 diabetes
54
2000
4
4.88
2.64
3 1.13 2.42 24.3 54.7 CAD
71
1000
4
5.37
3.13
3 0.96 3.56 6.31 27.8 51.8 Hyperlipidaemia
36
500
10
74.9
4.16
1.27 24.7 63 Hypertensive
63
2000
96
1.51 5.15 54.5
3.44
Baseline HDL-C (mmol/L) Baseline LDL-C (mmol/L) Baseline TG (mmol/L) Baseline TC (mmol/L) Baseline plasma concentration of vitamin C (mm/L) Duration of intervention (weeks) Vitamin C dose (mg/day) Male % BMI (kg/m2) Age (years) Health status Sample size Study design Author
Table 1 (continued )
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A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
Total score
4
facilitates the conversion of cholesterol into bile acids it may help lower blood cholesterol concentrations [10]. Another possible mechanism involves the action of vitamin C as an antioxidant. By inhibiting the oxidation of LDL-C, vitamin C facilitates its binding to, and uptake by, LDL receptors on hepatocytes (Fig. 1) [8]. Animals deficient in vitamin C exhibit hypercholesterolaemia and the subsequent administration of vitamin C may restore cholesterol concentrations to normal [10]. Furthermore, human observational studies showed that high plasma concentrations of vitamin C are associated with more favourable lipid profiles [5,11]. However, RCTs have produced contradictory results about the effect of vitamin C supplementation on blood lipids [12,13] and there is no evidence from RCTs that supplemental vitamin C reduces CVD risk [14]. A previous meta-analysis of 13 trials which recruited participants with hypercholesterolaemia showed that vitamin C supplementation reduced concentrations of LDL-C and triglycerides significantly with no significant effect on HDL-C concentration [8]. The present study aimed to conduct a systematic review and meta-analysis of RCTs investigating the effect of vitamin C supplementation on blood lipid (total cholesterol, LDL-C, HDL-C and triglycerides) concentrations. The secondary aim of the study was to determine whether the effects of such supplementation were modified by the participants' characteristics (health status, age, body mass index [BMI], baseline concentration of serum lipids and baseline concentration of vitamin C) or by features of the intervention (e.g. dose or duration of vitamin C administration). 2. Methods The present systematic review was conducted according to the Cochrane guidelines [15] and it is reported according to PRISMA guidelines [16]. 2.1. Literature search Four databases (PubMed, Embase, Scopus and Cochrane Library) were used to search for articles from inception until August 2014. In addition, a manual search of the reference lists of relevant reviews and articles included in the systematic review was performed. The following terms and keywords were used: vitamin C, ascorbic acid, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, triacylglycerol and randomised controlled trial. 2.2. Study selection The following criteria were applied to identify articles to be included in this systematic review and meta-analysis: 1) randomised controlled trials (no exclusion criteria were applied in relation to study design or blinding); 2) studies involving adults aged 18 years (no exclusion was applied for health status, smoking history or body size); 3) vitamin C administered alone, i.e. not combined with other drugs or nutritional interventions; 4) duration of intervention 2 weeks; 5) studies reporting changes in lipid profile (concentrations of total cholesterol, LDL-C, HDL-C and triglycerides); 6) English-language restriction but not time restriction was applied. Two investigators (FVV, NDW) independently screened the titles and abstracts of the articles to evaluate eligibility for inclusion. If consensus was reached, articles were either excluded or moved to the next stage (full-text). If consensus was not reached, the article was moved to the full-text stage and was appraised critically to determine eligibility for inclusion in the systematic review. Disagreements were resolved by discussion between the reviewers until consensus was reached.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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5
2.3. Data extraction and quality assessment
2.4. Statistical analysis
The following information was extracted independently by two investigators (AWA, FVV) from the eligible articles: 1) authors and year of publication; 2) study characteristics (design, sample size); 3) participant characteristics (age, male/female ratio, health status and baseline values for BMI, lipids concentration and plasma concentration of vitamin C) and 4) dose and duration of vitamin C intervention. Any disagreements in data extraction were resolved through discussion. In addition, the modified Jadad scale was adopted to assess the risk of bias of the included studies. Three main items: randomisation, blinding and description of dropouts or withdrawals were used to rate the quality of the included studies [17]. Possible scores ranged from 0 to 5 and a score of <3 indicates high risk while a score of 3 indicates low risk of bias.
The outcome of the meta-analysis is the net difference in lipids concentration (total cholesterol, LDL-C, HDL-C and triglycerides) at the end of the study between the intervention and control groups. Random effect models were used to take into account heterogeneity among studies. Forest plots were generated for graphical presentation of the serum lipid outcomes. Statistical analyses were performed using STATA 12 (StataCorp. 2011, College Station, TX, USA). The effect size was estimated as weighted mean differences (WMDs) with 95% confidence interval by using inverse variance weighting. Data not provided in the main text or tables were extracted from the figures. One study did not report the values for the standard deviations (SDs) [18]; the missing values were imputed according to the method described by Furukawa et al. [6]. Another study did not report blood lipids concentration at the end of
Fig. 3. Forest plot summarising the effect of vitamin C supplementation on total cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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A.W. Ashor et al. / Clinical Nutrition xxx (2015) 1e12
intervention for the placebo group [19]; in this case we substituted the baseline concentrations as the values for blood lipids at the end of intervention. For crossover trials, we used the mean and SD separately for the intervention and control conditions. This is regarded as a conservative approach that will reduce the power of these studies to show the true effect of the intervention [20]. In trials with multiple treatment arms and a single control group, the sample size of the control group was divided by the number of treatment groups to avoid over-inflation of the sample size [15]. Subgroup analyses were performed to identify possible sources of heterogeneity. The factors used in subgroup analyses included participant characteristics such as health status, age, baseline BMI, blood lipids and plasma concentration of vitamin C (median values). Further subgroup analyses were conducted according to study characteristics (dose, duration of vitamin C intervention and design and quality of the included studies). However, in this investigation we restricted subgroup analysis to cases with 5 studies per subgroup [15]. Meta-regression analysis was used to determine whether age, BMI, vitamin C dose, baseline concentration of serum lipids and of vitamin C influenced changes in the serum lipid profile in response to supplementation. Publication bias was evaluated by visual inspection of the funnel plot and by Egger's regression test [21]. Heterogeneity between studies was evaluated using Cochrane Q statistics; P > 0.1 indicates significant heterogeneity. The I2 test was also used to evaluate consistency between studies where a value <25% indicates low risk of heterogeneity, 25e75% indicates moderate risk of heterogeneity and >75% indicates high risk of heterogeneity [22].
3. Results 3.1. Search results The process of screening and selection of the studies is summarised in Fig. 2. The primary search of the four databases produced 1013 articles after the removal of duplicates. Additionally, three studies were found by manually searching the references of relevant reviews and studies. After title and abstract screening, 144 full-text papers were retrieved for further evaluation and a total of 40 publications were identified which met the inclusion criteria for entry into the systematic review. 3.2. Characteristics of the studies The total number of participants from 40 studies included in this systematic review was 1981 (1049 males, 932 females) with a median of 33 (range 8e305) participants per study. The overall median age was 52 (range 20e81) years. Trial designs included 29 parallel groups and 11 crossover studies. Two studies had two independent subgroups [23,24], therefore the total number of trials included was 42 (Table 1). Thirty-seven trials reported the effect of vitamin C supplementation on serum cholesterol, 35 on serum triglycerides, 29 on serum LDL-C and 35 on serum HDL-C concentrations. The health status of the participants in the trials differed; 12 trials included healthy individuals [11,18,19,25e33], 10 trials recruited diabetics [13,34e42], four recruited those with coronary artery disease [12,43e45], four trials recruited smokers [24,46e48],
Table 2 Pooled estimates of the effect of vitamin C supplementation on total cholesterol, LDL-C, HDL-C and triglycerides concentrations within various subgroups.
Age of participants (years)a 52 >52 Body mass index (kg/m2)a 26 >26 Baseline serum lipids (mmol/L)a TC 5.3; TG 1.7; LDL 3.4; HDL 1.16 TC > 5.3; TG > 1.7; LDL > 3.4; HDL > 1.16 Vitamin C dose (mg/day)a 1000 >1000 Plasma vitamin C concentration (mm/L) at baselinea 49.4 >49.4 Intervention duration (weeks)a 8 >8 Study Design Parallel Crossover Study quality (Jadad scale) Low risk > 3 High risk 3
Total cholesterol
LDL cholesterol
HDL cholesterol
Triglycerides
No. groups
No. groups
No. groups
No. groups
Effect size (95% CI) (P value) P ¼ 0.03
Effect size (95% CI) (P value) P ¼ 0.60
Effect size (95% CI) (P value) P ¼ 0.84
Effect size (95% CI) (P value) P ¼ 0.61
16 18
0.26 (0.45, 0.07) 0.03 (0.18, 0.13) P ¼ 0.58
15 12
0.13 (0.40, 0.13) 0.06 (0.39, 0.28) P ¼ 0.29
17 16
0.005 (0.05, 0.04) 0.001 (0.05, 0.05) P ¼ 0.27
18 15
0.04 (0.11, 0.03) 0.01 (0.23, 0.21) P ¼ 0.32
11 9
0.02 (0.12, 0.16) 0.13 (0.50, 0.23) P ¼ 0.06
11 7
0.13 (0.43, 0.17) 0.26 (0.41, 0.94) P ¼ 0.95
13 8
0.01 (0.05, 0.07) 0.07 (0.16, 0.01) P ¼ 0.78
12 8
0.04 (0.13, 0.20) 0.12 (0.37, 0.13) P ¼ 0.11
18
0.02 (0.12, 0.14)
16
0.06 (0.30, 0.19)
17
0.001 (0.04, 0.04)
17
0.05 (0.07, 0.18)
17
0.30 (0.51, 0.09)
12
0.10 (0.46, 0.26)
17
0.02 (0.04, 0.08)
16
0.14 (0.28, 0.002)
29 8
P ¼ 0.19 0.13 (0.26, 0.002) 0.01 (0.27, 0.25) P ¼ 0.46
26 3
P ¼ 0.07 0.15 (0.32, 0.02) 0.46 (0.24, 1.15) P ¼ 0.11
28 7
P ¼ 0.42 0.01 (0.02, 0.05) 0.03 (0.13, 0.07) P ¼ 0.06
28 7
P ¼ 0.26 0.06 (0.15, 0.03) 0.05 (0.26, 0.35) P ¼ 0.10
14 12
0.19 (0.30, 0.09) 0.17 (0.39, 0.04) P ¼ 0.61
19 18
0.08 (0.17, 0.20 (0.40, P ¼ 0.24 0.08 (0.23, 0.19 (0.29, P ¼ 0.10 0.06 (0.17, 0.36 (0.85,
25 12 30 7
8 9
0.34 (0.58, 0.09) 0.11 (0.31, 0.53) P ¼ 0.81
11 12
0.05 (0.02, 0.09) 0.03 (0.07, 0.01) P ¼ 0.82
12 12
0.14 (0.28, 0.002) 0.05 (0.12, 0.23) P ¼ 0.57
0.02) 0.01)
16 13
21 14
22 7
0.05) 0.13)
22 7
0.001 (0.05, 0.04) 0.01 (0.04, 0.06) P ¼ 0.57 0.00 (0.04, 0.04) 0.04 (0.004, 0.09) P ¼ 0.96 0.008 (0.02, 0.04) 0.03 (0.09, 0.15)
20 15
0.07) 0.08)
0.04 (0.23, 0.16) 0.15 (0.48, 0.18) P ¼ 0.27 0.03 (0.26, 0.20) 0.09 (0.28, 0.11) P ¼ 0.08 0.01 (0.21, 0.24) 0.41 (0.80, 0.02)
0.03 (0.13, 0.08) 0.08 (0.27, 0.12) P ¼ 0.06 0.03 (0.09, 0.14) 0.17 (0.32, 0.01) P ¼ 0.35 0.005 (0.12, 0.11) 0.12 (0.34, 0.10)
24 11 29 6
25 10 29 6
HDL, high-density lipoprotein; LDL, low-density lipoprotein. a Values sub-grouped by the median; P value for the differences between subgroups.
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7
three trials recruited patients with hyperlipidaemia [49e51], three trials recruited patients with chronic kidney disease [52e54] and there was one trial for each of the following: cerebrovascular disease [23], pre-hypertensive [55], hypertensive [56] and schizophrenic [57] participants. The duration of vitamin C supplementation ranged from two weeks to five years (median duration: eight weeks) and the dose of vitamin C ranged from 125 to 4500 mg per day (median dose: 1000 mg/day). The quality of the included studies ranged from 2 to 5 (median: 3) on the Jadad scale. On this scoring system, nine studies may have a high risk of bias [11,27,29,33,47,50,53,54,57] whereas the other 31 studies showed a score 3 on the Jadad scale (Table 1). Four studies described the method of randomization [12,26,34,35] and two studies stated the methods of allocation concealment [26,35]. For the crossover trials, most reported the washout period used (range 2e4 weeks) while three studies did not report this information [29,38,40]. Only one study reported the use of lipid-lowering agents during the intervention period by some of the included participants [12].
significantly greater effects of vitamin C supplementation in lowering total cholesterol concentration in those with higher baseline plasma cholesterol concentrations (b: 0.24, CI: 0.36, 0.11, P < 0.01) (Fig. 4A).
4. Meta-analysis
4.3. Effect of vitamin C on HDL-C concentration
4.1. Effect of vitamin C on total cholesterol concentration
Across all studies, vitamin C supplementation had no significant effect on HDL-C concentrations (0.004 mmol/L, 95% CI: 0.03, 0.03, P ¼ 0.83) and there was significant heterogeneity between studies (I2 ¼ 45; P < 0.01) (Fig. 6). However, supplementation improved HDL-C concentration in diabetics (0.06 mmol/L, 95% CI: 0.02, 0.11, P ¼ 0.01) (Supplemental Fig. 2 and Supplemental Table 3, “Supplementary data” in the online issue). Meta-regression analysis showed the change in HDL-C concentrations following vitamin C supplementation was greater in those with low baseline plasma concentrations of vitamin C (b: 0.002, CI: 0.003, 0.0001, P ¼ 0.04) (Fig. 4C).
Overall, vitamin C supplementation was ineffective in reducing total cholesterol concentration (0.10 mmol/L, 95% CI: 0.22, 0.01, P ¼ 0.08) and there was significant heterogeneity between studies (I2 ¼ 57.2; P < 0.01) (Fig. 3). However, subgroup analyses demonstrated a significant reduction in total cholesterol (0.26 mmol/L, 95% CI: 0.45, 0.07, P < 0.01) in younger participants (52 years age) following vitamin C supplementation (Table 2). Further information can be found in Supplemental Table 1 under “Supplementary data” in the online issue. Meta-regression analysis showed
4.2. Effect of vitamin C on LDL-C concentration Overall, there was no significant effect of vitamin C supplementation on serum LDL-C (0.09 mmol/L, 95% CI: 0.28, 0.11, P ¼ 0.39) and there was significant heterogeneity between studies (I2 ¼ 85; P < 0.01) (Fig. 5). However, subgroup analysis showed that vitamin C supplementation reduced LDL-C concentrations significantly in healthy participants (0.32 mmol/L, 95% CI: 0.57, 0.07, P ¼ 0.01) (Supplemental Fig. 1 and Supplemental Table 2, “Supplementary data” in the online issue). There was a trend towards a larger reduction in LDL-C concentration following vitamin C supplementation in participants with low baseline plasma concentrations of vitamin C (b: 0.01, CI: 0.004, 0.03, P ¼ 0.08) (Fig. 4B).
Fig. 4. Associations between baseline concentrations of serum lipids and of vitamin C and the effect of vitamin C supplementation on change in (A) total cholesterol, (B) low-density lipoprotein cholesterol (LDL-C), (C) high-density lipoprotein cholesterol (HDL-C) and (D) triglycerides concentrations. The size of each circle is proportional to the number of participants in that study.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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4.4. Effect of vitamin C on triglycerides concentration
5. Discussion
Overall, there was no significant effect of vitamin C supplementation on serum triglycerides concentration (0.03 mmol/L, 95% CI: 0.13, 0.08, P ¼ 0.62) and there was significant heterogeneity between studies (I2 ¼ 72.4; P < 0.01) (Fig. 7). However, in diabetics, vitamin C supplementation reduced serum triglycerides concentration significantly (0.15 mmol/L, 95% CI: 0.30, 0.002, P ¼ 0.01) (Supplemental Fig. 2 and Supplemental Table 4, “Supplementary data” in the online issue). In addition, meta-regression analysis demonstrated that vitamin C supplementation lowered triglycerides to a greater extent in those with higher triglycerides concentration at baseline (b: 0.17, CI: 0.30, 0.05, P ¼ 0.03) (Fig. 4D).
Overall, vitamin C supplementation had no significant effect on lipid profile. However, supplementation reduced total cholesterol concentration in younger (52 years) participants and LDL-C concentration in healthy participants. In addition, in diabetics, vitamin C supplementation reduced triglyceride and increased HDL-C concentrations significantly. Vitamin C produced bigger falls in total cholesterol and triglycerides in those participants with higher concentrations of these lipids at baseline and greater increase in HDL-C concentration in participants with low baseline plasma concentration of vitamin C. Despite the overall lack of effect of vitamin C supplementation on blood lipids concentration, this meta-analysis demonstrated that there may be specific groups within the population who would benefit from supplementation with vitamin C, including those with habitually low (baseline) vitamin C status or individuals at higher CVD risk (e.g. those with hypercholesterolaemia or dyslipidaemia). Therefore, research on identification of those with potential to benefit might offer an objective basis for individualising such interventions. For example, a previous study showed that supplementation with vitamin C reduced markers of oxidative stress in smokers with high BMI but no effect was seen in normal BMI smokers [58]. Similarly, Khan et al. found a significant improvement in endothelial function after vitamin C supplementation to healthy participants with habitually
4.5. Publication bias Visual inspection of the funnel plots showed no evidence of publication bias for the effect of vitamin C supplementation on total cholesterol (Supplemental Fig. 3A), LDL-C (Supplemental Fig. 3B), HDL-C (Supplemental Fig. 3C) or triglycerides concentration (Supplemental Fig. 3D, to be found online under “Supplementary data”). Egger's regression test outcomes (b: 0.03, P ¼ 0.43; b: 0.05, P ¼ 0.82; b: 0.26, P ¼ 0.16; b: 0.01, P ¼ 0.85 for total cholesterol, LDL-C, HDL-C and triglycerides respectively) confirmed the likely absence of publication bias for all four lipid fractions.
Fig. 5. Forest plot summarising the effect of vitamin C supplementation on low-density lipoprotein cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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9
Fig. 6. Forest plot summarising the effect of vitamin C supplementation on high-density lipoprotein cholesterol concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
low intakes of fruit and vegetables [59]. Furthermore, supplementation with multivitamins for six years in a population with high prevalence of micronutrient deficiency improved cerebrovascular disease mortality significantly [60]. These studies support the concept that individual vitamin status may determine the magnitude of the effect of antioxidant vitamin supplementation on CVD risk factors. In humans, a significant inverse association was found between plasma levels of vitamin C and risk of diabetes (OR ¼ 0.38, 95% CI: 0.28, 0.52) [61]. A recently conducted RCT demonstrated that administration of vitamin C to diabetic individuals resulted in significant reductions in fasting blood glucose and in glycosylated haemoglobin concentrations [62]. The possible mechanisms underlying the association of vitamin C with glucose metabolism include, i) the ability of vitamin C to prevent glycosylation of haemoglobin by inhibiting the formation of advanced glycosylation end products and ii) the ability of vitamin C to mitigate the longterm pancreatic b-cell dysfunction caused by oxidative stress [62]. The beneficial effects on triglyceride and HDL-C concentrations observed in the present meta-analysis might be explained by the ability of vitamin C to counteract the oxidative stress-induced insulin resistance in diabetic individuals. The vitamin C-induced
insulin sensitivity may, therefore, ameliorate the insulin resistanceinduced dyslipidaemia in diabetics (Fig. 1). In the present meta-analysis, a larger reduction in total cholesterol and triglycerides in response to vitamin C supplementation was observed in participants with higher concentrations of these lipids at baseline. This is in accord with a previous metaanalysis which showed significant reductions in LDL-C and triglycerides in individuals with hypercholesterolaemia [8]. Additionally, we observed greater reductions in LDL-C and increases in HDL-C following vitamin C supplementation in participants with lower plasma concentration of vitamin C at baseline. A previous meta-analysis showed that antioxidant vitamin supplementation improved arterial stiffness to a greater extent in individuals with low baseline plasma concentration of vitamin C [63]. Vitamin C supplementation did not alter total cholesterol significantly in older participants (>52 years). Vitamin C absorption may be less efficient in older individuals [64,65] and so higher doses of vitamin C might be required to produce significant beneficial effects on serum lipids. However, the relatively large doses of vitamin C (median 1000 mg/day) used in the studies included in this review make it unlikely that inadequate tissue availability explains the lack of effect of supplementation in older people.
Please cite this article in press as: Ashor AW, et al., Systematic review and meta-analysis of randomised controlled trials testing the effects of vitamin C supplementation on blood lipids, Clinical Nutrition (2015), http://dx.doi.org/10.1016/j.clnu.2015.05.021
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Fig. 7. Forest plot summarising the effect of vitamin C supplementation on triglycerides concentration. Squares indicate the effect size of each study summarized as weighted mean difference (WMD). The size of the shaded squares is proportional to the percentage weight of each study. Horizontal lines represent the 95% confidence interval.
A meta-analysis of 14 RCTs encompassing 90,056 participants showed that a mean reduction of 1 mmol in LDL-C is associated with a 23% reduction in myocardial infarction, a 17% reduction in stroke and a 12% reduction in all-cause mortality [66]. Using the outcomes from the present meta-analysis, this can be translated into possible reductions of 2.2%, 1.6% and 1.2% in myocardial infarction, stroke and all-cause mortality, respectively, with vitamin C supplementation. One of the major limitations of the present meta-analysis is the relatively small sample size of the included studies. However, most of the included studies used robust study designs and were rated as moderate/high quality on the Jadad scale. Another limitation is the small number of studies of participants with relevant medical conditions including coronary artery disease, hypertension and chronic kidney disease which limit our ability to draw conclusions regarding the effects of vitamin C supplementation in these conditions. Lastly, considerable heterogeneity was observed between studies which might be due to differences in study design and in the population groups included in these studies. Overall, this systematic review and meta-analysis found that vitamin C supplementation had no significant effect on lipid profile. This finding is in accord with the overall lack of effect of supplemental vitamin C on CVD outcomes observed in large-scale RCTs.
However, subgroup and sensitivity analyses showed that vitamin C supplementation reduced blood lipids significantly in participants with dyslipidaemia or low baseline vitamin C status. The results emphasise the potential importance of a personalised approach to vitamin C interventions in the primary and secondary prevention of CVDs. Future studies are required to confirm the observation that supplemental vitamin C appears to improve dyslipidaemia in individuals with type 2 diabetes and to explore the underlying mechanisms. Conflict of interest None. Acknowledgements AWA is funded by the Ministry of Higher Education and Scientific Research of Iraq. JCM acknowledges support from the LiveWell Programme a research project funded through a collaborative grant from the Lifelong Health and Wellbeing (LLHW) initiative, managed by the Medical Research Council (MRC) (grant number G0900686). The authors have no conflicts of interest to declare. The authors' responsibilities were as follows - AWA: drafted the manuscript;
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