Rachel , John McLaren , and Michael: Cardiovascular calcification and bone: A comparison of the effects of dietary and serum antioxidants.

Introduction

Cardiovascular (CV) calcification, often described as a form of sub-clinical atherosclerosis, can manifest as fully formed bone in arteries and heart valves when severe1,2. Atherosclerosis has long been thought to be, at least partially, caused by lipid oxidation and other pro-oxidants, with concomitant low endogenous antioxidant enzymes and low dietary intake of antioxidants. CV calcification is particularly prevalent in patients with type 2 diabetes and chronic kidney disease, largely due to increased oxidative stress and lipid oxidation products, as indicated by increased serum malondialdehyde3,4. Similarly, oxidative stress is also known to degrade bone and has been linked to osteoporosis, with malondialdehyde also being associated with osteoclastic activity. Several shared risk factors for CV disease and osteoporosis (smoking, diabetes mellitus, hypertension and inflammation) are associated with increased oxidative stress, while bone fracture itself can induce free radical generation5. Implicated in both the CV system and bone is nitric oxide (NO), which is associated with platelet aggregation and vascular relaxation, as well as modulating remodelling and stimulating bone loss; endothelial nitric oxide synthase (eNOS) is also widely expressed in bone6.

The main contributor to systemic oxidative stress is reactive oxygen species (ROS), reactive compounds that have one or more unpaired electrons, which can induce tumour necrosis factor-α (TNFα) and other pro-inflammatory cytokines. They may be generated by normal metabolism, ageing or oestrogen deficiency or may be produced in response to external stimuli, such as environmental toxins or inflammatory cytokines. While a certain level of ROS production is necessary for an effective immune response, overproduction induces an imbalance between cellular and tissue pro-oxidants and endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which may decline with age and lack of appropriate dietary nutrients.5,7 Antioxidants generally can inhibit expression of receptor activator of NF-kB ligand (RANKL)8,9, while vitamins C and E also act by increasing serum SOD and GSH10.

This review will discuss the effect of oxidative stress and dietary antioxidants (vitamins A, C and E, carotenoids, flavonoids, polyphenols, alpha-lipoic acid and N-acetylcysteine) on both CV calcification and bone. Although selenium, copper and zinc have proved effective in lowering osteoporosis and fracture risk, there are no human or animal studies showing their effect on CV calcification and they have therefore not been reviewed. An effect on bone parameters is taken to mean an effect on at least one skeletal site but not necessarily all sites.

Oxidative stress

CV calcification

Among the relatively few studies to have assessed the impact of oxidative stress on CV calcification, Ahmadi et al showed that in 60 asymptomatic subjects, there was a significant positive correlation between progression of coronary artery calcium (CAC) scores and vascular dysfunction and serum malondialdehyde11, while Watanabe et al showed that type 2 diabetics with aortic arch calcification had significantly higher levels of oxygen metabolites, which were more predictive of calcification than markers of inflammation12. Similarly, in induced animal aortic valve calcification, local superoxide and hydrogen peroxide were upregulated, with increased NADPH oxidase13, while in simulated uraemia and hypertension, oxidative stress induced aortic calcification and expression of osteoblastic proteins in VSMCs14,15, although there was no association between superoxide levels and aortic calcification16. Advanced glycation end products (AGEs), often found in diabetics, have been implicated in arterial calcification through their receptor (RAGE); induced calcification in rats could be reduced by inhibiting RAGE signalling17. Oxidative stress also increases osteoblastic differentiation and calcification in VSMCs18. ROS appear to act through increasing RANKL in VSMCs19, although lipid peroxidation, as indicated by increased malondialdehyde, is also common in VSMCs20,21 and the nitric oxide pathway may also be implicated6.

Although the case for oxidative stress as a cause of CV calcification is strong, the case for antioxidants as an inhibitor of calcification is not so clear. Valabhji et al found that the inverse association between total antioxidant status and CAC became non-significant after adjustment for age and/or diabetes duration22, while a SOD mimetic could reduce induced medial calcification in rats23 but worsened aortic valve calcification13. Nevertheless, antioxidants can generally inhibit RANKL expression8,9, while vitamins C and E also act by increasing serum SOD and GSH10 and selenium by increasing glutathione peroxidase levels25. Zinc can also raise insulin-like growth factor (IGF) 1, known to be involved in bone formation26, although in VSMC the driver for calcification may be IGF-227. Mice deficient in SOD manifested a weakness in bone stiffness and decreased BMD and reduction in the surface areas of osteoblasts and osteoclasts28.

Bone

Cross sectional28–30 and prospective studies28,31 clearly show that increased markers of oxidative stress and lower endogenous antioxidant enzymes are associated with lower BMD, osteoporosis, osteopenia and fracture incidence in older men and women. Systemic ROS increases and activates osteoclasts and promotes resorption via tumour necrosis factor (TNF) expression and nuclear factor kappa B (NF-kB) activation, through binding to its receptor RANKL, while inducible NOS (iNOS) is activated in osteoblasts by inflammatory cytokines to increase NO synthesis, which upregulates NF-kB production to stimulate bone resorption5,19,32. High RANKL is associated with lower BMD in men32. ROS also induce osteoblast apoptosis, inhibits osteoblastic differentiation and lower endogenous antioxidants7,28.

Vitamin A and carotenoids

Vitamin A is a fat-soluble vitamin, critical for vision, growth regulation and cell differentiation. It is ingested as either retinol (of animal origin) or carotenoids (of plant origin) and is dependent on adequate fat in the diet for absorption. Vitamin A is metabolized to active compounds, which are critical signalling molecules and can act through the binding and activation of a family of nuclear receptors expressed in VSMCs and endothelial cells.6,33 The conversion of carotenoids to vitamin A is feedback-regulated and ceases when adequate vitamin A is present33.

CV calcification

Among the few human studies of vitamin A intake and CV calcification or atherosclerosis, none has shown a correlation between dietary or serum vitamin A and CAC score34, carotid intima-media thickness (cIMT) or carotid plaque35,36. Nevertheless, retinoids, metabolically-active derivatives of vitamin A6 can effectively attenuate atherosclerotic vessel wall narrowing and improve vascular disease37. Furthermore, a study of menopausal women found a U-shaped association between serum retinol-binding protein (RBP)4 and CAC prevalence (with high and low levels giving higher CAC odds) but RBP4 was not correlated with cIMT or any CV risk factors38. Animal studies, however, showed that retinyl palmitate and retinoic acid treatment induce valve calcification, with increased expression of osteogenic genes39. There are no human carotenoid intake investigations and among the few serum studies there were no significant associations between incidence of calcified aortic plaque and serum lycopene, α-carotene, β-carotene, lutein or zeaxanthin levels, even in current or former smokers who are known to have increased oxidative stress. Two studies of plasma lycopene and cIMT showed mixed results in women but there was no association with serum alpha-carotene, beta-carotene, lutein or zeaxanthin35,36,40. Nevertheless, in the second and third quartiles of serum β-cryptoxanthin, an increased risk of aortic calcification was observed69, indicating an inverse U-shaped association.

Bone

Most cross-sectional41–44 and prospective43,45 studies found no relationship between dietary or supplemental vitamin A or retinol intake, serum concentrations and fracture risk or BMD, although a large 18 year study showed that those with a total vitamin A intake of ≥3000mcg/d had a significantly higher risk of hip fracture, which was primarily attributable to retinol46. Promislow et al suggested that the dose/response relationship between retinol intake and BMD is an inverse U-shape, with peak BMD occurring with a retinol intake of 600–840 mcg/d 47. There may also be an interaction with vitamin D, since there is competition for receptors; fracture risk was increased if vitamin A intake was ≥7508mcg/d (≥1426mcg/d retinol) compared to <5055mcg/d vitamin A (<474mcg/d retinol) if vitamin D intake was ≤11microg/d (≤440IU/d)41; similarly, in postmenopausal women with serum 25(OH)D <20ng/mL, the risk for osteoporosis increased substantially in women who had retinol blood levels of ≥80 μg/dL48,49.

Many carotenoid intake and serum studies have similarly shown no effect44–46,50 but among those that have showed an association, a large prospective study of the elderly found that those in the highest tertile of lycopene and β-carotene intake had a lower risk of hip fracture51, while higher intakes of β-carotene, lycopene and lutein/zeaxanthin were associated with less bone loss50. A large study by Wolf et al found that despite stratifying by calcium intake (threshold 500mg/d), of all antioxidant vitamins and minerals only total β-carotene intake was significantly associated with BMD, although there was no association with serum concentrations42. Increased dietary and serum beta-cryptoxanthin proved able to gamma-carboxylate osteocalcin in healthy individuals, a function normally reserved for vitamin K, increased bone calcium content and prevented bone loss in diabetic and ovariectomised rats52. A large intervention study gave either 25,000IU/d (7,576 mcg/d) retinyl palmitate for 10 years or 30mg/d β-carotene; there was no association between the cumulative intake of retinol and fracture risk but among men β-carotene was associated with a slightly reduced fracture risk53. A shorter study showed no effect at the same retinyl palmitate dose54. Mackinnon et al found that in healthy postmenopausal women, dietary lycopene restriction for one month resulted in decreased lutein/zeaxanthin and α- and β-carotene, with significantly increased N-telopeptides, with no effect on bone formation markers55.

Vitamin C

Vitamin C (or ascorbic acid), found in all fruits and vegetables, is a powerful antioxidant and an essential cofactor in enzymatic reactions, including several collagen synthesis reactions56–58. It has long been known as a preventer of scurvy59.

CV calcification

Ascorbic acid may, often together with alpha-tocopherol, lower induced calcification in VSMCs14,60–62, possibly by reducing transport of calcium ions61. In observational studies, however, intake of vitamin C was not associated with the CAC score in asymptomatic subjects34, while dietary and plasma vitamin C showed no association with cIMT or carotid plaque35. Intervention studies using a combination of treatments (a statin, ascorbic acid and alpha-tocopherol) reduced dyslipidaemia but had no effect on CAC score or progression or on incidence of CV events63, while ascorbic acid plus alpha-tocopherol reduced the ectopic calcification found in pseudoxanthoma elasticum64.

Bone

The majority of cross-sectional and prospective studies have found that higher vitamin C intake was significantly and positively associated with BMD45,51,65–67, with the benefit apparently deriving from long term supplementation (mean intake 745mg/d)51,57, although a large study found no association except in women on hormone therapy, where vitamin C enhanced the effect42. Where vitamin C intake in postmenopausal women was stratified by calcium intake, it was found that all the increase in BMD was clustered among those with calcium intake >500mg/d while there was no association between vitamin C intake and BMD where calcium intake was lower58. Leveille et al found that food-derived vitamin C was not associated with BMD in postmenopausal women but supplements for women aged55–64, but not older, used for ≥ 10 years showed a significantly higher BMD68.

Blood vitamin C was inversely correlated with fracture risk in elderly adults69,70 and was significantly lower in elderly osteoporotic females29. Some studies found that low vitamin C status in smokers significantly increased the risk of hip fracture, particularly where vitamin E intake was low66,71, although others have found no association72. Intervention studies show mixed results, which may be due to trial length. Postmenopausal women receiving 10g/d oral vitamin C showed no effect on markers of bone turnover in a four week study73, although 1000mg/d given with 600mg/d vitamin E for six months showed a significant improvement in spine BMD74 and 500mg/d vitamin C in combination with 400mg/d vitamin E for 90 days increased SOD, GSH and osteoblastic markers in osteoporosis patients10. Although humans have lost the ability to synthesis vitamin C, knock-out studies in animals show that vitamin C deficiency correlated with increased fracture risk, decreased expression of bone formation markers75 and increased osteoclastogenesis by increasing expression of RANK76, while treatment generally reduced bone fragility and improved healing, BMD and osteoblastic survival28,77, while reducing lipid peroxidation and osteoclast differentiation, with decreasing NF-kappaB and RANKL expression78.

Vitamin E

Vitamin E collectively refers to the ten fat-soluble, chemically distinct isoforms: α, β, γ, δ and ε tocopherols (saturated) and tocotrienols (unsaturated)79. The commercial availability of vitamin E is mostly in the form of α-tocopherol, which has the highest biological activity of all isomers and is the most abundant form of vitamin E in human tissues and serum, as it is selectively retained in the body80. The principal function of vitamin E is to act as an antioxidant and is thought principally to protect against lipid peroxidation but it also exhibits a powerful anti-inflammatory effect with its ability to inhibit cyclooxygenase-2 (COX-2) and cytokine production and decrease C reactive protein32, although this is not seen in every study81. Vitamin E is found in vegetable oils, green leafy vegetables, nuts, seeds and egg yolks.

CV calcification

Two cross-sectional studies showed that in healthy subjects aged 39-45 and older haemodialysis patients, there was no correlation between the CAC score34 or peripheral arterial calcification score82 and dietary vitamin E but when supplemental intake (likely α-tocopherol) was also assessed, those supplementing had a significantly higher risk of CAC than those not supplementing (105.5mg/d vs 76.4mg/d)34. Nevertheless, in middle-aged women, increasing intake of vitamin E was associated with a progressive decrease in cIMT35 and carotid plaque36. These results are reflected in a large intervention study of adults aged 50-70 with CAC scores at or above the 80th percentile for age and gender, showing that a combination of a statin, vitamin C and 1,000IU/d α-tocopherol for mean 4.3 years lowered dyslipidaemia but had no effect on CAC progression63. It seems probable that α-tocopherol can be protective against CAC in conditions of oxidative stress, such as CKD, but is ineffective or detrimental where vitamin E levels are adequate; few studies test baseline or end-point status.

Bone

A 2012 systematic review of vitamin E supplementation found that in three epidemiological studies of women, none showed any association between total vitamin E intake and BMD changes, although among the eight animal studies, seven showed positive changes in bone structure or biochemistry, with tocotrienols giving better results compared to α-tocopherol80. Among the human studies, Maggio et al found that in women with osteoporosis, plasma levels of vitamin E were significantly lower29 and a more recent investigation showed that in early postmenopausal women, a lower α-tocopherol/lipid ratio was associated with greater risk of osteoporosis and lower BMD83. Despite this, Macdonald et al showed that increased dietary intake of vitamin E was inversely associated with BMD although the addition of intake from supplements removed this association67 and a number of smaller studies have shown a detrimental association between vitamin E intake or serum levels and BMD or fracture risk, particularly among those with high oxidative stress, such as smokers31,71,72. In the only two human studies to consider other vitamin E isomers, Hamidi et al showed that high serum γ-tocopherol and a low ratio of serum α-tocopherol to γ-tocopherol were associated with increased bone specific alkaline phosphatase, a marker for bone formation, although there was no association with urinary N-telopeptides, a marker for bone resorption84, while Wolf et al found no association with BMD for serum α- or γ-tocopherol42. Intervention studies mostly involved a combination of vitamin E and vitamin C and generally showed a significant benefit over no supplementation in postmenopausal women74, although animal studies suggested that the effect of α-tocopherol is inferior when compared to vitamin C81. It also appears that when α-tocopherol levels are above normal range it may antagonise vitamin K, necessary for healthy bone formation85. An additional reason for the inconsistent results may be that vitamin E displays a U-shaped dose-response curve.

Animal studies have also shown that osteoporosis, lower bone calcium content and poor mechanical strength could be prevented by a mixture of α-tocopherol, α-tocotrienol, γ-tocotrienol and δ-tocotrienol, which reduced PGE2, pro-inflammatory cytokine and lipid peroxidation levels, possibly through down-regulation of RANKL expression, and raised endogenous antioxidant enzymes. Tocotrienols may also act by activating the oestrogen receptor. Although α-tocopherol alone had no effect, and may be detrimental in excess, possibly through inhibition of the action of tocotrienols, γ-tocotrienol showed considerable promise for bone health.86 Other studies have confirmed that α-tocopherol may promote fracture healing, through its ability to raise bone SOD and GPX and serum CAT concentrations81.

Flavonoids and polyphenols

Flavonoids and polyphenols are substances known to have powerful antioxidant effects, particularly those found in tea and coffee, such as epigallocatechin gallate (EGCG), rutin and quercetin, and resveratrol, found in red wine, while the barley and hops used to make beer are also rich polyphenol sources.

CV calcification

Two large studies of older adults investigated coffee intake but showed mixed results, with one finding that coffee drinking was independently correlated with aortic calcification presence87, while another showed that >3 daily cups of coffee significantly reduced incidence of a CAC score of >40088. Among the tea and coffee polyphenols and flavonoids, EGCG from green tea reduced renal calcification in rats89, while quercetin attenuated VSMC calcification independently of matrix Gla protein through inhibition of transglutaminase 2 (TG2) and β-catenin signalling90–92 and reduced calcification of porcine heart valves93,94 and the rat aorta in CKD, although catechin and rutin were also partially effective95. Human studies of alcohol intake show mixed results, which may be due to a J-shaped association and different effects according to alcohol type [96,97], although most studies show a protective effect of moderate alcohol consumption on CHD risk98. Few studies stratify by type of alcohol but nevertheless, resveratrol supplementation reduced aortic calcification and atherosclerotic lesions in CKD mice and monkeys fed a high fat and sucrose diet through activation of sirtuin 1 (SIRT1), a histone deacetylase, which was significantly downregulated in phosphate-induced calcification99–101. In vitro studies have found that the polyphenols in wine and beer were able to concentration-dependently inhibit VSMC alkaline phosphatase activity91,102.

Bone

Tea consumption was associated with reduced fracture risk among older women in southern Europe; the fact that most subjects drank black tea suggests that the benefit is independent of any added milk103. Both black tea104 and green tea105 were generally associated with increased BMD among older women but Vestergaard et al suggest that the benefit from tea may be due to lower coffee consumption106, which was found in some studies to result in lower BMC and higher fracture risk in those drinking more than two cups a day107,108, although others have found no effect109. To the extent that caffeine is detrimental, it may be due to its potential to reduce calcium absorption and increase its urinary excretion, although this has been contradicted in other studies; provided calcium intake exceeds 800mg/d, moderate caffeine is unlikely to be detrimental to bone. Tea catechins have been shown to inhibit bone resorption, increase osteoblast proliferation and, at high doses, decrease osteoclast generation in vitro, while increasing BMD and BMC in rats. Similar effects have also been found with the tea flavonoids rutin and quercetin. Although tea contains caffeine, levels are relatively low.109 Alcoholism has often been associated with osteoporosis but other studies have shown a protective effect, with a J-shaped dose/response curve with 1-2 drinks per day correlating with improved BMD in postmenopausal women; when stratified by type of alcohol, wine gave the greatest benefit in women and beer in men, while spirits gave the lowest BMD110. In vitro studies have also shown that resveratrol is active in osteoblastic MC3T3-E1 cells and may have a synergistic effect with vitamin D in promoting bone health111.

Alpha-lipoic acid

Αlpha-lipoic acid (ALA) is made in the human body and is essential for aerobic metabolism and as a cofactor in several enzymatic reactions. It is present in all foods but particularly offal, spinach, broccoli and yeast extract.

CV calcification

There are no human studies of the effect of ALA on CV calcification but in mice with calcification from heart tissue injury, ALA lowered tissue damage and reduced calcification112, while in rabbits it reduced aortic valve and medial calcification13,113.

Bone

In the only human study, 44 osteopenic postmenopausal women were given ALA combined with vitamin C, vitamin E, and selenium for one year; the improvement in BMD barely achieved statistical significance114. Nevertheless, in animal studies, ALA administration increased BMD levels and decreased inflammatory markers and RANKL and down-regulated genes associated with bone resorption and apoptosis and up-regulated those associated with bone formation and the IGF-1 signalling pathway115–117.

N-acetylcysteine

N-acetylcysteine (NAC) functions principally as a mucolytic agent and helps to increase endogenous glutathione levels. It is not found in food sources but is available as a dietary supplement

CV calcification

There are no human studies of NAC and CV calcification but in mice with oxidative stress-induced aortic calcification, NAC slowed the rapid progression of atherosclerotic lesions and reversed the increase in plaque collagen content [118], while in hypertensive uraemic rats, NAC suppressed markers of cellular senescence associated with arterial calcification [15]. In vitro studies showed that NAC could inhibit osteoblast apoptosis and calcification and reduce osteopontin production in VSMCs119,120.

Bone

There are no human studies of NAC and bone but in animal studies it raised tissue glutathione levels in female rats77, abolished a reduction in bone formation, so increasing BMD and BMC121 and increased bone healing122. In vitro studies confirm that NAC can prevent osteoclast formation, and can down-regulate ROS generation and inflammatory mediators (NF-kB, RANKL, TNF-alpha and IL-6) in osteoblasts, reduce apoptosis and dose-dependently increase intracellular glutathione77,123. Additionally, NAC can inhibit homocysteine-induced oxidative stress and bone resorption124, while increasing alkaline phosphatase and matrix mineralisation, with up-regulation of bone-related genes122.

Discussion

There is a significant association between oxidative stress, in the form of oxidised lipids, ROS and the presence and progression of CV calcification, which is likely mediated by increased inflammation. Nevertheless, the association with endogenous antioxidant enzymes is less clear, although they can be increased by dietary antioxidants. In bone, however, there is a consistent correlation of bone loss and fracture with elevated markers of oxidative stress and reduced antioxidant enzymes.

Dietary antioxidants vary considerably in their effect on CV calcification and bone. Vitamin A and most carotenoids show little association with calcification, CVD or bone, although high retinol and β-cryptoxanthin intake may be detrimental, while lycopene and β-carotene may be beneficial for bone. The apparent lack of association may be due to vitamin A and carotenoids displaying a U-shaped dose/response curve or competition for attachment to vitamin D receptors. By contrast, the associations for vitamin C are much stronger in bone, where the beneficial effect occurs with long-term supplementation of mean intake 745mg/d and calcium intake ≥500mg/d, but there seems to be little association on CV calcification. Smokers, who have high markers of oxidative stress and risk of calcification and bone loss, are particularly at risk of low vitamin C.

The effect of vitamin E is more difficult to assess because so many of the studies investigate α-tocopherol, which may be a risk factor for CV calcification and fracture in high intake, while ignoring other isomers. Nevertheless, in conditions of oxidative stress, such as renal failure, it may be beneficial. Furthermore, where beneficial results are achieved with supplementation with α-tocopherol and vitamin C, it seems the benefit derives largely from vitamin C. In bone, γ-tocopherol and tocotrienols show promise but the number of studies is few. Overall, it appears that α-tocopherol is only beneficial in conditions of high oxidative stress, such as renal disease, smoking or fracture healing. Studies of other substances with antioxidant properties are extremely limited for CV calcification, particularly human studies, and for flavonols and polyphenols it has been necessary in some cases to use the beverages that contain them. Although there are also mixed results for coffee, resveratrol EGCG and quercetin appear to be particularly effective in both the CV system and bone. Similarly, ALA and NAC appear effective in lowering calcification risk presence and improving bone parameters in animals.

There are a number of potential reasons for the lack of clear association between antioxidant intake and serum levels and CV calcification and bone. Firstly, the association may not be linear. Secondly, there may only be benefit in condition of oxidative stress, when both dietary antioxidants and endogenous antioxidant enzymes are depleted, but where there is no deficiency, an increase in antioxidants has only marginal utility. Unfortunately the effect of antioxidants is rarely stratified by baseline serum levels. Thirdly, there may be an interaction with other bone vitamins, with dietary lycopene restriction reducing lutein/zeaxanthin and α- and β-carotene absorption. There is also an increased fracture risk with high vitamin A and low vitamin D intake or serum levels and the possibility that high intake of alpha-tocopherol may antagonise vitamin K-dependent proteins, although increased dietary and serum beta-cryptoxanthin proved able to gamma-carboxylate the vitamin K-dependent protein osteocalcin in healthy individuals, Fourthly there may be an interaction with calcium intake; increased vitamin C intake improved BMD only where calcium intake was >500mg/d, Finally, studies may be testing the wrong isomer of vitamin E; high serum α-tocopherol was associated with lower BMD, while a low ratio of serum α-tocopherol to γ-tocopherol was associated with increased bone formation. Similarly, α-tocotrienol and γ-tocopherol have shown promise in in vitro studies8,24 but have not been tested on humans, although α-tocopherol was shown to suppress bioavailability of other vitamin E isoforms125–127.

Conclusion

This review of the effects of oxidative stress and antioxidants has shown a consistent association of measures of oxidised lipids and ROS and CV calcification and bone, which may be mediated through increased inflammation. Endogenous antioxidant enzyme concentrations, although clearly correlated in bone, are less obviously associated in the CV system. A survey of dietary antioxidants and their serum levels shows that some have no effect on CV health or bone, while β-cryptoxanthin and high levels of α-tocopherol may be a risk factor for CV calcification, while high retinol intake and possibly α-tocopherol were associated with increased fracture incidence. One potential reason for this is that vitamin A, carotenoids and α-tocopherol are all fat-soluble vitamins, which may show a U-shaped dose-response curve, but since baseline serum levels are rarely tested it is not possible to determine whether the subjects began the study already replete, in which case further supplementation would either have no effect or be detrimental. Water-soluble vitamin C, however, has proved beneficial in bone, where it shows more of a linear dose-response curve, although there is little effect on the CV system but studies are few. Similarly, although little studied, tocotrienols, tocopherols (with the exception of α-tocopherol), resveratrol, EGCG, quercetin, ALA and NAC may be effective in both the CV system and bone. In general, few studies include an overview of the general nutrient intake of participants. Micronutrient interdependencies as well as macronutrient balance may strongly influence nutrient uptake and metabolism. The inclusion of baseline levels and a nutritional overview could increase the relevance of future studies.

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