1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References

Abstract:  Vitamin C is a pivotal redox modulater in many biological reactions of which several remain poorly understood. Naturally, vitamin C has been the subject of many investigations over the past decades in relation to its possible beneficial effects on cardiovascular disease primarily based on its powerful yet general antioxidant properties. However, growing epidemiological, clinical and experimental evidence now suggests a more specific role of ascorbate in vasomotion and in the prevention of atherosclerosis. For example, in contrast to most other biological antioxidants, administration of vitamin C can apparently induce vasodilation. Millions of people worldwide can be diagnosed with vitamin C deficiency according to accepted definitions. In this perspective, the present review examines the evidence for a specific link between vitamin C deficiency and increased risk of atherosclerosis as well as the possible mechanisms by which vitamin C may exert its protective function.

In recent years, there has been growing evidence that vitamin C may have a more specific role in the protection of atherosclerosis than merely its contribution to the maintenance of the basic redox homeostasis of the body. Thus, in vitro evidence suggests that vitamin C is closely involved in maintaining the bioavailability of nitric oxide, which is the pivotal signalling molecule in the vasculature mediating relaxation of the vascular smooth muscle cells and protecting against endothelial dysfunction [1,2].

Most mammals are capable of synthesizing their own ascorbate and are thus not prone to develop vitamin C deficiency. However, humans have a mutation in the gene encoding gulonolactone oxidase – the rate limiting enzyme in ascorbic acid synthesis; hence, they rely on obtaining adequate amounts of vitamin C through the diet. Although scurvy is now considered a rare disease – at least in the Western world – epidemiological studies suggest that large subpopulations (between 5% and 30% depending on socioeconomic status, smoking status, etc.) can be diagnosed with hypovitaminosis C [3–5], defined as a plasma concentration <23 µmol/l [6].

Interestingly, several of the risk factors associated with the development of cardiovascular disease have also been shown to correlate with low plasma levels of vitamin C in population studies. These include, for example, smoking [7], ageing [8], diabetes [9], obesity [10] and hypercholesterolaemia [11]. Moreover, plasma vitamin C levels have been shown to correlate inversely with prevalence of cardiovascular disease in a number of studies when corrected for confounding factors [12–14].

To further investigate the suggested role of vitamin C in protection against atherogenesis in more detail, it is important to have in vivo models in which the ability to develop atherosclerosis can be combined with the long-term marginal vitamin C deficiency that is characteristic in human subpopulations. The guinea pig is an obvious candidate for such models as this species is among the few that, like humans, do not synthesize ascorbic acid (in contrast to rats, mice and rabbits etc.) and apparently is susceptible to the development of diet-induced atherosclerosis.

In the present review, the evidence supporting an atherogenic effect of marginal vitamin C deficiency and its specific role as cofactor in the endothelial production of nitric oxide is examined with emphasis on guinea pigs as in vivo model as well as from a human perspective.

Vitamin C and dyslipidaemia in guinea pigs

  1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References

Early studies.

Guinea pigs have been the primary in vivo model of vitamin C deficiency since the 1930s [15,16] and some 20 years later, it was shown that vitamin C deficiency on its own could cause atherosclerotic lesions in the aorta of guinea pigs. These studies were conducted by Willis [17,18] and laid the foundation for later investigations of the effect of ascorbic acid on cholesterol metabolism [19–21]. In particular, Ginter et al. recognized in a series of studies that deficiency of ascorbic acid affected the cholesterol metabolism resulting in an inverse relationship between vitamin C and the cholesterol levels in blood and liver [20,22–24]. An important aspect of this is the transformation of cholesterol to bile acids which takes place in the liver. Vitamin C deficiency causes the activity of the rate-limiting enzyme in conversion of cholesterol to bile (7α-hydroxylase) to decrease; hence, the decreasing excretion of cholesterol leads to hypercholesterolaemia [25,26]. As cholesterol feeding was well known to induce changes in the aorta of rabbits, it was soon applied to guinea pigs in combination with low vitamin C levels [26].

The guinea pig as in vivo model of vitamin C deficiency.

In table 1, a list of studies performed on guinea pigs with the purpose of evaluating dyslipidaemia and/or atherosclerosis is given. Animals were fed diets low in vitamin C or high in cholesterol and/or fat and compared to control animals on normal feed, whereas in some studies a combination of dietary regimens was used (references in table 1). Generally, focus has been on end-points of known relevance to atherosclerosis such as amount of lipoproteins and lipids in the blood as well as actual presence of atherogenic changes (plaques or fat deposits in the vasculature). Also, changes in the metabolism of cholesterol have been considered relevant end-points as they affect the lipoprotein profile.

Table 1.  A listing of guinea pig studies where the effect of vitamin C on factors involved in atherogenesis has been investigated.
ReferenceReported signs of scurvyDuration of experimentNumber of guinea pigs per groupPlasma vitamin C levels (µM)End-pointsEffect of vitamin C deficiency alone (regular diet)Effect of low ascorbic acid when an atherogenic/high cholesterol diet is also given, as compared to atherogenic/high cholesterol diet with sufficient vitamin C
  • TC, total cholesterol; PL, phospholipids; TG, triglycerides; AA, vitamin C; chol, cholesterol; FC, free cholesterol; EC, esterified cholesterol.

  • *

    Starved for 16 hrs before samples were obtained.

17+12–41 days8–32Plasma cholesterol[UPWARDS ARROW]
Presence of atherosclerotic changes in aorta[UPWARDS ARROW][UPWARDS ARROW]
18+21–42 days12–27Presence of atherosclerotic changes in aorta[UPWARDS ARROW]
26 10 or 20 days respectively10– (tissue concentration measured)Serum lipidsTC[RIGHTWARDS ARROW], PL[RIGHTWARDS ARROW]
24 112–143 days12– (tissue concentration measured)Serum cholesterol[UPWARDS ARROW]
Serum fatty acid composition[RIGHTWARDS ARROW]
27 139–142 days6–14– (tissue concentration measured)Serum total cholesterol[RIGHTWARDS ARROW]
28+Up to 150 days?Presence of atherosclerotic changes in aorta[UPWARDS ARROW]
Lipoprotein lipase activity in aorta and liverAorta[RIGHTWARDS ARROW]Liver[UPWARDS ARROW]Aorta[RIGHTWARDS ARROW]Liver[UPWARDS ARROW]
22 17 weeks11–12Serum lipidsTC[UPWARDS ARROW], TG[UPWARDS ARROW]
30 6–9 weeks15Low AA/high cholesterol: 20 ± 6SDPlasma lipidsTC[RIGHTWARDS ARROW], TG[UPWARDS ARROW]
High AA/high cholesterol: 79 ± 14
Histology of aorta (severity of atheromatous changes)Some changeMost change
Lipid stained area of aorta [UPWARDS ARROW]
32 16 weeks5–6– (liver concentration measured)Serum lipidsTC[UPWARDS ARROW]
LDL turnover ratesT1/2[UPWARDS ARROW], fractional catabolic rate[DOWNWARDS ARROW]
Low AA/no cholesterol: 9.1 ± 3.4
High AA/high cholesterol: 28 ± 4.5
Low AA/high cholesterol: 5.7 ± 2.3
Low AA/no cholesterol: 42 ± 6.3
High AA/high cholesterol: 60 ± 8.5
Low AA/high cholesterol: 28 ± 4.5
Atheromatous change in aorta [UPWARDS ARROW]
35 5 weeks8Fatty streak/plaque formation in aorta[UPWARDS ARROW]
37 4 weeks5High AA/no cholesterol (non-starved): approximately 36Serum lipids/lipoproteinsTC[RIGHTWARDS ARROW], TG[RIGHTWARDS ARROW], HDL[RIGHTWARDS ARROW], LDL[RIGHTWARDS ARROW], VLDL[RIGHTWARDS ARROW]
Low AA/no cholesterol (non-starved): approximately 11
High AA/no cholesterol (starved*): approximately 12
Low AA/no cholesterol (starved): approximately 3
LDL susceptibility to oxidation[UPWARDS ARROW]
LDL kineticsLDL apoB flux rate[UPWARDS ARROW], apoB pool size[RIGHTWARDS ARROW], LDL fractional catabolic rate[RIGHTWARDS ARROW]
Hepatic HMG CoA reductase and ACAT activityHMG CoA reductase [RIGHTWARDS ARROW]ACAT[UPWARDS ARROW]
LDL receptor in liver[DOWNWARDS ARROW]
Hepatic HMG CoA reductase and ACAT activityHMG CoA reductase activity[DOWNWARDS ARROW] ACAT[UPWARDS ARROW]
TG and apoB secretion rates TG secretion rate[RIGHTWARDS ARROW] apoB secretion rate[UPWARDS ARROW]
Size of VLDL particles[DOWNWARDS ARROW]

A highly relevant issue when interpreting and extrapolating results from these in vivo model studies is whether the very low plasma concentrations of ascorbic acid in the models reflect actual vitamin C levels in humans. In the third National Health and Nutrition Examination Survey (NHANES III), it was found that approximately 10% of the investigated American population suffered from severe vitamin C deficiency (defined as plasma concentrations <11 µM) and that even more (approximately 20% of males and 15% of females) had marginal deficiency (in this case defined as 11–28 µM) [4], which is not considered to be an immediate risk of scurvy but still is classified as deficiency [6]. In a Scottish population highly ranked for cardiovascular disease and considered to belong to the low socioeconomic-status group, it was found that 44% of the population had plasma vitamin C levels <22.7 µM and even 20% was <11.3 µM [3]. The plasma levels measured in the reported guinea pig studies were >36 µM in the control groups (up to 86 µM) whereas the vitamin C deficient groups had concentrations ranging from 3–20 µM (table 1). Accordingly, the values fit well with the concentrations observed in humans with vitamin C deficiency in the above-mentioned population studies.

Vitamin C deficiency versus clinical scurvy.

A major pitfall in in vivo studies of vitamin C deficiency is the often unclear distinction between chronic suboptimal vitamin C status and the development of clinical scurvy. According to the major epidemiological studies, a sizeable minority of population of the Western world can be characterised as being chronically vitamin C deficient while cases of clinical scurvy are rarely reported [40,41]. Vast metabolic changes take place during scurvy that are not commonly found in the Western population situation regardless of their vitamin C status, and these may therefore cause inconsistent experimental findings and hamper the ability to draw conclusions based on animal experiments with regard to the involvement of vitamin C deficiency in atherogenesis in humans. Consequently, in vivo models of acute scurvy should not be considered relevant for the study of atherogenesis in humans [42]. For this reason, information of scurvy or scurvy-like symptoms is included in table 1. In some of the reported studies, animals were not kept on their respective diets for longer periods of time because of signs of or actual death from scurvy, demonstrating that the vitamin C administration was so low that the conclusions cannot distinguish between effects of chronic vitamin C deficiency or clinical scurvy.

The guinea pig as in vivo model of dyslipidaemia.

Finally, it should be considered if the characteristics of the dyslipidaemia displayed by guinea pigs are comparable to those of human dyslipidaemia. Fernandez et al. have thoroughly investigated this issue in a number of papers [43–45]. They conclude that for several cases such as cholesterol distribution, activity of enzymes involved in lipoprotein metabolism, LDL receptor binding domain characteristics, and cholesterol synthesis etc., the guinea pigs share human features and therefore this species is recommended as an animal model for studying dyslipidaemia in vivo.

Effect of vitamin C deficiency on lipid metabolism

  1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References


As can be seen from tables 1 and 2, the majority of the guinea pig studies has used changes in blood cholesterol as endpoint, and most authors have found that chronic vitamin C deficiency causes the levels of total cholesterol to rise irrespective of whether the diet is atherogenic or not. The same pattern is seen with the levels of the lipoproteins, LDL and VLDL. The effect seems to be more pronounced when an atherogenic diet is given in combination with a vitamin C deficient diet. The levels of HDL generally decrease in vitamin C deficiency: again with a more prominent effect when the diet is also atherogenic.

Table 2.  A quantitative overview of the publications cited in table 1.
End-pointEffect of vitamin C deficiency alone (regular diet)Effect of low ascorbic acid when an atherogenic/high cholesterol diet is also given, as compared to atherogenic/high cholesterol diet alone
  1. [UPWARDS ARROW] represents an increase, [RIGHTWARDS ARROW] represents no change and [DOWNWARDS ARROW] represents a decrease of the given parameter. The numbers in the parentheses represent the actual number of papers that report the given change.


In the study by Sharma et al., no change was found in any of the measured lipoproteins as a result of a low vitamin C diet [34]. However, the vitamin C dose given in this study was higher than in the other studies. Thus, the plasma vitamin C concentration was about 40 µM in the normal diet group with low vitamin C as compared to approximately 30 µM in the atherogenic marginal vitamin C diet group, that is, several fold the levels representing severe vitamin C deficiency in humans [6]. As the intention of the above study was to test if high doses of ascorbic acid had a beneficial effect on the outcome obtained with the atherogenic diet – which they found – and not to keep the animals on as little vitamin C as possible, this does not conflict the findings by most other authors. In one conflicting study by Nambisan and Kurup [29], the total cholesterol levels were unaffected by keeping the guinea pigs on very low levels of vitamin C, yet the combination of vitamin C deficiency with a diet high in cholesterol exacerbated the effects seen with high cholesterol alone. Also, other authors found no effect of vitamin C deficiency on plasma cholesterol [26,27,30,37]. However, two of the latter were short-term experiments and may not have provided enough time for manifestation of the changes seen in the remaining studies [26,37].


Triglycerides have been found to constitute an independent risk factor for atherosclerotic disease [46] and potential mechanisms are related to the triglycerides being found in the lipoproteins chylomicrons and VLDL [47]. The composition of VLDL is very similar between man and guinea pig [48,49]. Once the triglycerides are removed by lipoprotein lipase, the remaining remnant has the ability to penetrate the arterial wall and cause lipid infiltration which is considered a hallmark of early atherosclerosis [50,51].

From the literature given in table 1, there is no clear relationship between vitamin C and triglyceride status. Thus, about an equal number of studies show either that low vitamin C with an otherwise non-atherogenic diet causes the triglyceride levels to rise or find no effect of poor vitamin C status, respectively. However, when an atherogenic diet is given in combination with the low vitamin C levels, all studies including analysis of triglycerides found that the levels increased, which could possibly exert a pro-atherogenic effect on the vascular wall as judged by the human studies referred to above.

The mechanism behind vitamin C affecting the triglyceride concentrations may involve suppression of carnitine levels as it has been shown that vitamin C deficiency causes the amounts of carnitine in guinea pigs to decrease [52]. As this cofactor is responsible for transporting fatty acids into the mitochondria, this could lead to a decreased clearance of triglyceride from the blood subsequently resulting in hypertriglyceridaemia [53,54].


In many of the guinea pig studies, phospholipid determination has been part of the validation of the degree of dyslipidaemia, since cholesterol, triglycerides and phospholipids collectively constitutes the major lipids found in blood. However, as the term phospholipid covers a vast amount of compounds highly involved in cellular signalling and inflammation, conclusions on these findings may be ambiguous. In spite of this possible heterogeneity, it appears from tables 1 and 2 that phospholipids tend to increase in vitamin C deficiency.

Atherogenic changes in aorta.

In studies, where the actual presence of pathological atherosclerotic changes in the aorta has been evaluated, they all find a correlation between low vitamin C status and atherosclerotic progression.

In most studies where lipids were extracted from the aorta, it was generally found that all types of lipids (total cholesterol, triglycerides and phospolipids) increased with vitamin C deficiency [26,29,31]. In contrast, one experiment [34] found that the levels of phospolipids decreased with low vitamin C intake when the diet was also atherogenic, yet in agreement with the above, most of the remaining lipids in this study were increased with vitamin C deficiency.


A few of the more recent guinea pig studies have looked closer into lipoprotein homeostasis by studying composition and metabolism of some of these particles. In particular, LDL has been investigated in detail [38,39].

Fernandez et al. found that vitamin C deficiency did not change the structure of LDL except for an increase in the amount of free cholesterol (non-esterified) in the particles [38]. Also the ability of Cu2+ incubation to induce lipid oxidation was investigated, and here they found that low plasma concentrations of vitamin C doubled thiobarbituric acid reactive substance (TBARS) formation, a biomarker of lipid hydroperoxides, but only if the animals were also fed a diet rich in polyunsaturated fats.

In the study by Montano et al. [39], it was found that plasma cholesteryl ester transfer protein (CETP) activity was increased which could account for some of the increase of LDL cholesterol and decrease in HDL cholesterol as this enzyme is responsible for transferring cholesteryl esters from HDL to LDL. The reported changes in liver enzymes (reduced HMG CoA reductase activity and increased Acyl-CoA cholesterol acyl transferase (ACAT) activity) could result in increased cholesterol accumulation in the liver as HMG CoA reductase is responsible for turning cholesterol into bile acids. This increases the substrate availability for ACAT esterifying-free cholesterol for storage which is in line with the finding that hepatic cholesteryl ester levels were positively correlated to the ACAT activity. Also hepatic LDL receptor levels were found to be lower, hence decreasing the clearance of LDL by this organ.

The overall conclusion from these two studies [38,39] is that vitamin C deficiency affects lipoprotein homeostasis such that the blood cholesterol composition is shifted towards a more atherogenic profile.

A few earlier studies also looked into lipoprotein metabolism [29,32]. Ginter and Jurcovicova found that the turnover of LDL was decreased [32]. This was reflected by both an increase in the half-life of this lipoprotein as well as a decrease in the fractional catabolic rate. Again, these findings implicate that shortage of vitamin C causes pro-atherogenic changes by increasing LDL levels. Nambisan and Kurup studied lipoprotein lipase activity and found that hepatic but not aortic levels were increased [29]. Only low levels of lipoprotein lipase activity were found in guinea pig liver as compared to other guinea pig tissues, and the localization of this enzyme was mainly in the endothelial cells in the periportal areas. Moreover, very little mRNA is detectable in guinea pig liver by in situ hybridization [55], hence supporting the finding that lipoprotein lipase in the liver is taken up from the blood [56]. It has been debated whether lipoprotein lipase exerts anti- or pro-atherogenic effects [57]. Consequently, the relevance of increased levels of lipoprotein lipase activity in the liver remains unclear.

Vitamin C and atherogenesis – beyond dyslipidaemia

  1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References

It is commonly accepted that the function of the endothelial cell layer plays an essential role in the development of atherosclerosis. In the early stages of atherogenesis, the endothelial cells become dysfunctional which is closely related to a reduction in the bioavailability of nitric oxide. This initiates a cascade of pre-atherogenic events such as reduced vasodilatation, formation of pro-inflammatory mediators as well as it causes activation of platelets, hence rendering the vasculature in a pro-thrombotic state [1,2].

A widely used method for assessing endothelial function in vivo is by endothelium-dependent vasodilatation [58], and numerous studies have been undertaken to investigate the effect of vitamin C in this setting, yet the majority of these studies have taken place in humans whereas data from animal experiments are sparse.

In humans, the effect of vitamin C on endothelium-dependent vasodilatation has been examined in a variety of disease states such as obesity, hyperhomocysteinaemia, diabetes, renal disease, coronary artery disease, etc. The conclusions from these studies are presented in table 3. The majority of the studies have investigated how acute vitamin C administration (e.g. intravenous, intraarterial or high oral dose given a few hours before the experiment) affects endothelial-dependent vasodilatation. The vast majority of studies found that vitamin C caused the vasodilatation to improve. When it comes to long-term administration, for days and up to months, the results are more conflicting as no effects were found in more than half of the experiments (referenced in table 3).

Table 3.  Clinical studies evaluating endothelium dependent vasodilatation. Acute administration of vitamin C covers intra venous and intra arterial infusion, as well as a high dose given per-orally on the day of the experiment. Long-term administration covers vitamin C tablets given for days, weeks and months before the experiments were performed. Only studies examining the effect of vitamin C on a single condition have been included.
Condition associated with endothelial dysfunctionAcute vitamin C improved dilatationLong-term vitamin C improved dilatationNo effect by acute vitamin CNo effect by long-term vitamin C
Ageing59,60 6161
Diabetes (also acute hyperglycaemia and hyperinsulinaemia included)62–6869 70–73
Coronary artery disease74–83   
Hypercholesterolaemia84–87 8888
Hyperlipidaemia (acute)95–99   
Metabolic syndrome116,117   
Other cardiac diagnosis79,120–125126,127128 
Renal disease129–131 130 
Sedentary lifestyle132–134  132
Pharmacologically induced endothelial dysfunction147,148   

Another important aspect to pay attention to is that in those studies reporting positive effects exerted by vitamin C, the healthy/unaffected controls did not show any benefit [59,60,62–64,66,74,76,79,82,84,85,87,89–91,95,101,103,105–109,115,118–121,124,132–136,138,142,143,147,149–151]. Only in one study [83], it was reported that vitamin C also improved the condition of the otherwise healthy non-smokers.

In studies performed in animals (referenced in table 4), ascorbic acid generally also has the ability to improve the vasodilatory response when the animals are in a non-healthy state and the vasodilator acts via endothelium-dependent mechanisms [152–160]. When stimulating the vasculature by non-endothelium-dependent vasodilators, no effect was found from vitamin C in these studies.

Table 4.  In vivo animal studies (other than guinea pig) in which endothelial dysfunction has been evaluated after administration of vitamin C.
ReferenceSpeciesAntioxidant therapyConditionAscorbate in plasma (µM)End pointEffect of vitamin C/antioxidants in response to
152PigAA 1 g/day and vitamin E 100 IU/dayNormal (no pathology induced)Control: 98.1 ± 3.1Myocardial perfusionAdenosine: [DOWNWARDS ARROW]
Antioxidant: 180.3 ± 8.4 Dobutamine: [DOWNWARDS ARROW]
     Epicardial vasorelaxationBradykinin: [UPWARDS ARROW]
Substance P: [UPWARDS ARROW]
153PigAA 3 g/dayBalloon-induced ED of LADControl: –LAD blood flow in response to acetylcholine (Ach), 5-HT, adenosine, and isosorbide dinitrate (ISDN)Acetylcholine (Ach)[UPWARDS ARROW]
Control + ED: 4.9 ± 1.7Serotonin (5-HT): [UPWARDS ARROW]
Vitamin C + ED: 72.4 ± 7.6Isosorbide dinitrate (ISDN): [RIGHTWARDS ARROW]
155PigAA 1 g/day and vitamin E 100 IU/kgRenovascular disease (RVD) induced by hypercholesterolaemia and renal artery stenosisControl: 2.3 ± 0.6Renal blood flow (RBF)Ach: [UPWARDS ARROW]
RVD: 1.7 ± 0.6Sodium nitroprusside (SNP): [RIGHTWARDS ARROW]
Antioxidant + RVD: 4.0±0.6
154PigAA 1 g/day and vitamin E 100 IU/kgRenovascular disease (RVD) induced by hypercholesterolaemia and renal artery stenosisControl: 2.3 ± 0.6RBFAch: [UPWARDS ARROW]
RVD: 2.3 ± 0.6
Antioxidant + RVD: 3.7 ± 0.6
156PigAA 1 g/day and vitamin E 100 IU/kgRenovascular hypertension (HT) induced by a stent in the left renal arteryControl: 2.5 ± 0.6Myocardial perfusionAdenosine: [UPWARDS ARROW]
HT: 3.2 ± 0.4Dobutamine: [RIGHTWARDS ARROW]
HT + antioxidants: 5.3 ± 0.4
157PigAA 1 g/day and vitamin E 100 IU/kgDiet-induced hypercholesterolaemiaRBFAch: [UPWARDS ARROW]
104PigAA 1 g/day and vitamin E 1000 IU/dayDiet-induced hypercholesterolaemiaIliac artery flowAch: [UPWARDS ARROW]
Nitroglycerin: [RIGHTWARDS ARROW]
159PigAA 1 g/day and vitamin E 100 IU/dayDiet-induced hypercholesterolaemiaControl: 97 ± 5.6Myocardial perfusionAdenosine: [UPWARDS ARROW]
Hypercholesterolaemic: 84.8 ± 2.0
Hypercholesterolaemic + antioxidants:132.6 ± 7.9
166PigAA 1 g/day and vitamin E 100 IU/kgDiet-induced hypercholesterolaemiaControl: 95.8 ± 8.1RBFAch: [UPWARDS ARROW]
Hypercholesterolaemic: 86.8 ± 2.7
Hypercholesterolaemic + antioxidants: 133.3 ± 10.1

Possible mechanisms for the vasodilatory actions of vitamin C.

The overall findings from the human and animal studies given above point to some important aspects of the mechanisms by which vitamin C acts. First of all, the reason for apparent discrepancies between healthy and non-healthy subjects could be that the positive effect of vitamin C administration is related to the change from hypovitaminosis C to normal vitamin C status rather than from normal to super saturated status. This is in agreement with the general findings that low levels of vitamin C correlates with several of the risk factors for cardiovascular disease [5,9,10] that are also known to decrease the function of the endothelium [5,9,10,161–163]. Moreover, vitamin C displays highly dose and concentration-dependent absorption kinetics suggesting that little if any effect can be expected, if the subjects are saturated at entry [5]. Secondly, vitamin C must exert its effect through the endothelium as only vasodilators acting via endothelium-dependent mechanisms can have their effect improved [152–156,158]. Finally, the response is able to act immediately as also direct administration of vitamin C into the blood (either i.v. or intra arterially) has an effect [52,59,60,62–67,75–78,80,81,84–88,91,95–98,100–110,115–125,129,132–136,138,142,143,147,148,150,151,179].

It is tempting to speculate that nitric oxide directly could be a target for this recovery of vasodilatation, as this signalling molecule is fast acting and also highly prone to oxidation. Especially, the oxidation of nitric oxide by inline image, which results in the formation of peroxynitrite, is very likely to take place. It has been described as the only reaction able to outcompete the reaction rates of the endogenous enzyme intended to remove this free radical (superoxide dismutase), due to the amounts of nitric oxide produced and the fast reactivity [164].

When it comes to the antioxidant actions of vitamin C, a listing of the thermodynamic properties of endogenous redox active molecules places ascorbate (being >99% deprotonated at physiological pH) in a very high ranking concerning the ability to act as a reductant under physiological conditions [165]. Thus, vitamin C appears to have the ability to reduce nitric oxide in vivo. However, this rationale has been questioned in a study by Jackson et al. who reported that physiological levels (even up to the mM range, which are achievable intracellularly [166]) of ascorbate are too low to protect nitric oxide from forming peroxynitrite upon superoxide anion generation as validated by 3-nitrotyrosine formation, hence other mechanisms by which vitamin C can increase the bioavailability of nitric oxide must exist [167].

It has been shown that ascorbate can increase the activity of endothelial nitric oxide synthase in vitro and thus the production of nitric oxide [168–170]. To produce nitric oxide, endothelial nitric oxide synthase is dependent on several cofactors: NADPH, FAD, FMN, haem and tetrahydrobioptherin (BH4). These molecules enable electrons to be transferred within the active site of endothelial nitric oxide synthase resulting in the net formation of l-citrulline and nitric oxide from l-arginine and O2 [171,172].

A plausible link between vitamin C and production of nitric oxide seems to be BH4. The specific role of BH4 is to stabilize the nitric oxide synthase dimer [173,174] as well as to act as a donor of electrons from haem to l-arginine [175,176]. If BH4 is oxidized, it will no longer bind to endothelial nitric oxide synthase which will then uncouple and produce superoxide anion rather than nitric oxide (for a thorough review see [164]). Several studies have investigated the effect of BH4 administration and found that modulating systemic BH4 affects the vasculature in both humans and animals [177–183]. Heller et al. reported in vitro evidence that vitamin C may have a specific role as a redox modulator in nitric oxide production as they found that vitamin C increases intracellular BH4 concentrations as well as the BH4 half-life in solution [184].

Taken together, these data provide a rationale for the possible negative influence of vitamin C deficiency on the vasculature (fig. 1). Hence, certain disease states creating imbalances in redox homeostasis or unhealthy living (smoking, lack of fruit and vegetable intake etc.) cause the vitamin C levels to decrease [7,185,186], which affects the reduction state of BH4 and results in a decreased nitric oxide bioavailability. This renders the vasculature in a pro-atherogenic state that together with the dyslipidaemia – which is also exacerbated by deficiency of vitamin C – it promotes the development of cardiovascular disease. This hypothesis is well in line with the correlation between risk factors for cardiovascular disease and low plasma vitamin C status reported in epidemiological studies [9,10,187].


Figure 1. Summary of the potential involvement of vitamin C deficiency in atherogenesis. (A) Ascorbate plays an important role in keeping tetrahydrobiopterin (BH4) reduced. Lack of vitamin C impairs the reduction of the BH3-radical (inline image) to BH4 and renders its oxidation to dihydrobiopterin (BH2) the preferred reaction. BH2 cannot bind to endothelial nitric oxide synthase (eNOS) and the result is eNOS uncoupling. This causes eNOS to produce superoxide rather than nitric oxide. Superoxide has a high affinity for nitric oxide and their reaction forms the deleterious compound peroxynitrite (ONOO). The combined effect of these events is endothelial dysfunction. (B) Vitamin C is involved in the conversion of cholesterol to bile acids via the 7α-hydroxylase in the liver. In vitamin C deficiency, less cholesterol will be excreted by this mechanism causing the levels of circulating cholesterol to rise. (C) Vitamin C is a cofactor in the biosynthesis of carnitine. Lack of this cofactor decreases the carnitine-mediated transport of triglycerides into the mitochondria (mTG = mitochondrial triglyceride) resulting in a increasingly pro-atherogenic lipoprotein profile. (D) Ascorbate is a powerful antioxidant placed low in the antioxidant hierarchy and may be important for the maintenance of redox balance in the blood stream. Thus, oxidative stress in the vasculature may increase directly by lack of vitamin C. This increase in oxidative stress can both induce endothelial dysfunction and also increase the formation of oxLDL. The combined effect of the resulting redox imbalance is believed to promote atherogenesis.

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To our knowledge, there have not yet been reports from animal studies designed to elaborate specifically on this hypothesis in vivo as the present studies have used animals that do not rely on obtaining their vitamin C through the diet as they possess the ability of endogenous synthesis. As can be seen from the information given in table 3, only in a single study [153], the plasma vitamin C levels are comparable to what is considered to be saturated and deficient levels in humans [6,188] (72.4 ± 7.6 µM for the control group and 4.9 ± 1.7 µM for the injured group). In this study, endothelial function was evaluated by stimulated vasodilatation in pigs after balloon-induced endothelial damage in a coronary artery. The authors concluded that chronic vitamin C supplementation could reduce the decrease in vasodilatation resulting from endothelial damage. In the remaining in vivo animal studies given in table 3, plasma vitamin C levels were either higher than the approximately 70–80 µM, which is considered the saturation steady-state plasma level in humans [188], or the plasma concentrations were below 10 µM in both experimental and control groups.

None of these scenarios reflect how endothelial function is affected by replenishing depleted plasma levels of vitamin C in humans and it is thus questionable if the pig can be used as a model animal for vitamin C supplementation.

Yet, the use of genetically modified mice could address these issues in the future. A mouse strain lacking gulonolactone oxidase activity has been produced and even crossed with apolipoprotein E knockout mice, which are very frequently used for atherosclerosis research. Nakata and Maeda have used this double knock out approach [189] to investigate how vitamin C deficiency for several months affected atherosclerotic plaque formation. They were able to keep the plasma vitamin C at approximately 10 µM in the deficient groups; however, this decrease did not affect the amount of plasma cholesterol as compared to control mice irrespective of the diet being high in cholesterol or not. The main findings of this study were that lack of vitamin C did not seem to affect lesion number or size whereas there were significant changes in the collagen content of the plaques. Hence, they speculated that lack of vitamin C might cause the plaques to become more prone of rupture.

The use of mice genetically engineered to both, lack the ability to produce ascorbic acid as well as to become susceptible to the development of atherosclerosis could be a possible model for replacing the guinea pig. Great care should be taken to avoid overruling the effects of vitamin C by excessive plaque formation.

To our knowledge, there are not yet any data on endothelial dysfunction measured in vivo in mice and rats, but with the increasing possibilities offered by the knock out technology this could be a possible approach for future studies.


  1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References

From the studies investigating the effect of vitamin C on blood lipid levels in guinea pigs, the overall finding is that low plasma levels of vitamin C have the ability to induce dyslipidaemia and atherogenic changes – especially if an atherogenic diet high in cholesterol and/or fat is given simultaneously.

To further investigate the mechanism by which vitamin C deficiency affects the vasculature as suggested from the findings in human population studies, animal models are needed. In this respect, the guinea pig has two major advantages over other laboratory animals in general. Like humans, they are not capable of synthesizing endogenous vitamin C, and atherogenic changes can be induced by dietary regimes. Certain genetically modified mouse strains also fulfil these criteria and might therefore also be of use for further elaboration on vitamin C deficiency and endothelial dysfunction.

So far, the experiments performed in guinea pigs have mainly focused on dyslipidaemia, hence future experiments are needed to further characterize the possible role of ascorbate as redox modulator in the biosynthesis of nitric oxide in vivo.


  1. Top of page
  2. Abstract
  3. Vitamin C and dyslipidaemia in guinea pigs
  4. Effect of vitamin C deficiency on lipid metabolism
  5. Vitamin C and atherogenesis – beyond dyslipidaemia
  6. Conclusion
  7. References
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