The Multiple Effects of Vitamin D against Chronic Diseases: From Reduction of Lipid Peroxidation to Updated Evidence from Clinical Studies

 

 

Vitamin D (Vit D) is a fat-soluble vitamin represented by two related fat-soluble substances, cholecalciferol (Vit D3) and ergocalciferol (Vit D2), both of which can be used to cure or prevent rickets. These molecules are produced from 7-dehydrocholesterol, known as Pro-Vit D, which is activated to Vitamin D by ultraviolet light, usually in the dermis or epidermis.

 

These sterols are then transported to the liver, where they undergo 25-hydroxylation (to 25-OH Vit D), and then to the kidneys, where they undergo a second hydroxylation to the fully active molecules: 1,25 dihydroxycholecalciferol (calcitriol) and 1,25-dihydroxyergocalciferol. Humans synthesize sufficient amounts of Vitamin D when they have adequate exposure to sunlight.

 

Moreover, Vitamin D acts more as a hormone than a vitamin, binding to specific cytosolic receptors, mainly located in intestinal epithelial cells and osteocytes, as well as in numerous other tissues, including hematopoietic cells, hair follicles, adipose tissue, muscle, and the brain. After binding to its cytosolic receptors, 1,25(OH)2D is transported to the nucleus, where the vitamin-receptor complex interacts with DNA and modulates gene expression to increase calcium uptake.

 

The most important effects of Vitamin D are calcium absorption and bone resorption; however, it exerts many other effects, not all of whose clinical implications are yet fully known.

 

Vitamin D is involved in many physiological processes and plays a therapeutic role in human diseases, such as cancer, infectious diseases, osteoarticular, and cardiovascular diseases. There are many conflicting data on the role of Vitamin D in both prevention and therapeutic effects in many diseases, especially heart disease and cancer. In general, conventional doses of Vitamin D are well tolerated, with no significant adverse effects.

 

High doses of Vitamin D can be toxic and lead to a number of signs and symptoms, but not to liver damage or jaundice. Unfortunately, the lack of rigorous clinical studies makes it difficult to draw clear conclusions about the therapeutic aspect of Vitamin D, especially in cancer patients. There are several sources from which Vitamin D can be obtained, such as food, supplements, or the skin (photosynthesis). Apart from fortified foods and fish (especially fatty fish), Vitamin D content in foods is generally not very high. Vitamin D occurs in various forms known mainly as Vit D3 (cholecalciferol) and Vit D2 (ergocalciferol).

 

Vitamin D is synthesized in the skin by exposure to ultraviolet (UV) radiation from the sun on its precursor, 7-dehydrocholesterol. Vitamin D2 (ergocalciferol) is another form of Vitamin D that is often used for fortification. In fungi (e.g., mushrooms), Vitamin D2 can also be produced by UVB irradiation of ergosterol, its precursor. Dietary sources of Vit D include fish, especially in the form of oil (Vitamin D3), egg yolk (Vitamin D3), and mushrooms (Vitamin D2), as well as artificially fortified cereal products and dairy products, such as milk (Vitamin D2 or Vitamin D3).

 

As mentioned above, the production of Vit D3 in the skin is independent of the involvement of enzymes and occurs via 7-dehydrocholesterol (7-DHC). In short, UV light in the spectral range of 290–320 nm UVB (under sunlight) can lead to the formation of pre-vitamin D3, which is isomerized to Vitamin D3 in a thermosensitive but non-catalytic process.

 

The rate of D3 formation depends on both UVB intensity and the degree of skin pigmentation. This process can be affected by skin melanin, clothing, and sunscreens, which can prevent UVB from reaching the 7-DHC and, thus, negatively affect Vitamin D3 production. The mechanism of action of Vit D can be discussed in genomic and non-genomic terms. The genomic aspect has been studied in detail by Pike et al. and Haussler et al.. In the genomic mode of action, the vitamin D receptor (VDR) plays a key role.

 

The vitamin D receptor is encoded by the VDR gene. VDR belongs to the superfamily of nuclear hormone receptors, which are ligand-induced transcription factors. It is worth noting that this receptor also functions as a receptor for the secondary bile acid, lithocholic acid.

 

It consists of three main domains: first, a DNA-binding domain characterized by two zinc fingers that are able to bind specific DNA sequences (called VDRE); second, a C-terminal ligand-binding domain; and third, a specific region that is able to connect both domains. Notably, VDR interacts with the retinoid X receptor. Interestingly, VDR binds to VDRE, forming a complex that differs from cell to cell by forming the action specificity of Vit D and that can regulate the expression of several genes, including C/EBPα and others.

 

In summary, the following aspects can be considered as the basis for the effect of VDR/RXR on target genomes:
(a) the number of binding sites for the Vit D receptor depends specifically on the cell type;
(b) the VDR/RXR heterodimer is considered the major active transcription unit, but not the only one;
(c) the VDR binding sites are mainly (but not exclusively) classical hexamer half-sites separated by three base pairs;
(d) the enhancers of the VDR-encoding gene are located near or far, i.e., promoter proximally or promoter distally, and many enhancers are also located in clusters hundreds of kilobases away from their target genes;
(e) enhancers are modular, and the binding sites they possess are binding sites for a number of different transcription factors;
(f) these enhancers (populating a genome) are particularly dynamic and cell-type-specific.

 

Vit-D also has a rapid effect on selected cells that is probably unrelated to gene regulation and appears to be mediated by another receptor, which is potentially membrane-bound. One of the most important non-genomic effects of Vit D is the stimulation of calcium and phosphate uptake from the small intestine, known as trans-calcification. This term was coined by Norman’s laboratory to describe the rapid onset of calcium flow through the intestine of a Vit-D-fortified chick fed with 1,25(OH)2D. Moreover, Vit D has been shown to modulate not only calcium, but also chloride channel activity, protein kinase C activation and distribution, and phospholipase C activity in various types of cells, including osteoblast, liver, muscle, and intestinal cells. Vit D can also stimulate phosphate reabsorption in renal tubules and trigger the secretion of calcium from the bones into the blood.

 

Another significant effect of Vit D administration is the reduction in pro-oxidative biomarkers and lipid peroxidation; in fact, in patients with type 2 diabetes, Vit D administration reduces glucose-related pro-inflammatory proteins and 4-hydroxinonenal (a lipid-peroxidation marker involved in cardiovascular diseases, cancer, and neurodegenerative diseases). Notably, Vit D plays a key role in the homeostasis of neuron functions in humans. Recent meta-analyses correlate 25OHD serum levels with a high risk of cognitive impairment and memory decline. Other studies correlate low 25OHD serum levels with the initiation and progression of Parkinson’s disease.

 

The conclusions of a meta-analysis on Vit D and neurodegenerative diseases declare that Vit D at 75 nmol/l could be useful to improve bone health, cognitive functions, and neuron survival through reductions in oxidative stress and improvements in mitochondrial metabolism.

 

Vitamin D and Osteoporosis

Vit D3 promotes the absorption of dietary calcium via parathyroid hormone (PTH), contributing to adequate calcium homeostasis. The activity of 25(OH)D-1α-hydroxylase, the enzyme responsible for the conversion of 25(OH)D to 1,25(OH)2D, is stimulated by PTH and inhibited by 1,25(OH)2D itself.

 

Furthermore, Vit D3 suppresses the activity of PTH and its secretion by inhibiting the proliferation of parathyroid cells. This active calcium absorption occurs through the induction of the synthesis of a protein expressed at the brush borders of intestinal epithelial cells, which bind the ion and transport it from the lumen to the cell cytoplasm. In addition, 1,25(OH)2D also facilitates the passive absorption of calcium by increasing the permeability of intercellular “tight junctions”.

 

Vit D deficiency results in a compensatory increase in PTH, which in turn stimulates bone turnover and renal tubular reabsorption of calcium to maintain calcium homeostasis. Calcium and phosphorus are deposited in the collagen matrix and form hydroxyapatite, which gives strength to the skeleton. Through these mechanisms, Vit D acts indirectly on the bones. Vit D deficiency leads to hypocalcemia and hypophosphatemia, which classically cause rickets in children and osteomalacia in adults. Both diseases are caused by impaired bone mineralization due to an inadequate calcium-phosphate product and the effect of PTH on the kidneys, causing phosphaturia.

 

Adequate levels of Vit D have an important effect on bone mass in young and old people. Since bone mineral density directly correlates with fracture risk, Vit D is crucial for the treatment of osteoporosis. During life, the skeleton is subject to different processes, which can be termed modeling and remodeling. Modeling aims to adapt the structures of the bones to the mechanical stresses caused by body growth and age. Remodeling continues throughout life, with the aim of replacing damaged or aged bone tissue with new tissue, without altering the affected surface, ultimately controlling bone structure and function. The cells that regulate these processes are osteoblasts, osteoclasts, osteocytes, and lining cells.

 

Osteoblasts originate in multi-potential mesenchymal progenitors and are exclusively responsible for the formation, deposition, and mineralization of bone tissue. They also control the recruitment, differentiation, and maturation of osteoclasts, which participate in resorption activity. In addition, osteoclasts associated with bone resorption also express several factors that regulate osteoblast function [100]. Osteocytes, which are terminally differentiated osteoblasts, act as mechano-sensors and modulate both osteoblast and osteoclast activity. Similarly, bone lining cells play a role in assisting osteoclasts and osteoblasts in bone remodeling.

 

The discovery of osteoprotegerin (OPG), a receptor activator of nuclear factor kappa-B ligand (RANKL) derived from osteoblasts, led to a better understanding of the mechanism of cross-communication between osteoblasts and osteoclasts. RANKL is a transmembrane protein on the surface of osteoblast cells that binds to its own receptor, RANK, which is located on the surfaces of both osteoclast precursors and mature osteoclasts. This cell-to-cell contact, in combination with m-CSF, which is also produced by osteoblasts, stimulates the differentiation of precursor cells into osteoclasts and increases their activity. 1,25(OH)2D regulates this process via the VDR receptor expressed by the osteoblast by inducing RANKL.

 

Osteoporosis is a clinical condition characterized by a progressive reduction in bone mass and a concomitant alteration in the skeletal microarchitecture, leading to a loss of bone strength, which increases the risk of fractures due to very mild trauma. Osteoporotic fractures mainly occur at the level of the vertebral bodies and the femoral neck. There are essentially two forms of the disease: primary and secondary osteoporosis.

 

 

Vitamin D and Muscle Homeostasis

Low levels of Vit D are associated with an increased risk of falls and proximal weakness. Muscle pain and hypotonia are typical clinical features of rickets and osteomalacia. Proximal myopathy with unsteady gait is observed in adults with severe chronic Vit D deficiency. Observational studies have reported an association between low Vit D levels and the risk of falls in the elderly. 1,25(OH)2D stimulates muscle protein production and contributes to muscle contraction by regulating the mechanism of calcium transport at the level of the sarcoplasmic reticulum.

 

Age-related sarcopenia is associated with an increased risk of falls, disability, and mortality in the elderly. VDR’s expression in skeletal muscle suggests that Vit D may act directly on this tissue. Experimental studies in skeletal muscle-specific VDR knockout mice suggested a Vit D-specific effect on muscle fiber size, as a reduction in muscle fiber diameter of the II types was observed in these animals. This provides biological evidence for a direct role of Vit D deficiency in sarcopenia. This is also supported by another study speculating the possibility that Vit D modulates myostatin, a negative regulator of muscle mass.

 

During aging, Vit D synthesis in the skin mediated by UV radiation decreases, as well as the renal activation of 25OHD and VDR concentrations in muscle tissue. The combination of all of these effects could make the muscle more susceptible to Vit D deficiency and promote falls.

 

Adequate sunlight exposure is the most cost-effective method to maintain Vit D levels because its natural sources in food are very limited.

 

It has been calculated that whole-body sun exposure in summer provides the equivalent of 10,000 IU Vit D. Studies have demonstrated that, on average, 25OHD levels increase by 0.5 to 1 ng/mL for every 100 IU of Vit D supplement administered. Much higher doses are likely to be required in obese individuals or those with malabsorption (including after bariatric surgery). People with darker skin, older age, and higher BMI are more likely to be affected by Vit D deficiency.

 

 

Vitamin D Supplementation and Modulation of Immune Functions

Vit D is able to modulate immune functions in humans. Both 25(OH)D and 1,25(OH)2D act in multiple ways in several immune cells, such as macrophages, monocytes, B, and T-type lymphocytes. Vit D is able to modulate the expression of the genes involved in innate and adaptative immune functions. In fact, epidemiological studies correlate autoimmune diseases and the risk of infection with low serum levels of 25OHD. However, interventional studies aimed at improving the serum levels of 25OHD in patients with immune-related diseases are heterogeneous and not always significant. Historical evidence for the links between Vit-D and innate immunity comes from reports dating from the mid-18th to the early 19th century, before the antibiotics era when Vit-D3-rich cod liver oil and sunlight exposure were used to treat tuberculosis. Humans have a precise system of regulation for their immune activities based on the endogenous hyperactivation of Vit D.

 

During an infection process, activated polymorphonuclear granulocytes, through pro-inflammatory chemokines and growth factors, stimulate CYP27B1, which is able to convert 25OHD to 1,25(OH)2D, which, through autocrine mechanisms, increases the expression of cathelicidin. Cathelicidin exerts antiviral and antibacterial effects on Mycobacterium tuberculosis and others. Moreover, 1,25(OH)2D acts in a paracrine manner. This stimulates adjacent macrophages, which often appear in the bloodstream, reaching serum concentrations of 30 ng/mL, causing hypercalcemia (hypercalcemia is also a marker of infectious status).

 

Moreover, 1,25(OH)2D is able to maintain immune tolerance in APC cells and finely manage the surface expression of MHC class II, immunogenic cytokines, and co-stimulation molecules. The modulation of the immunogenic cytokine profile is a key orchestrator of immune homeostasis; for example, Vit D increases IL-10 expression, which is characterized by anti-inflammatory activity.

 

Conversely, 1,25(OH)2D reduces the expression of the pro-inflammatory and atherogenic cytokines involved in immune hyperactivation, such as IL-6 and IL-17. Notably, 1,25 (OH)2D is able to modulate the metabolism and immunogenic activity of NK cells; although the clinical studies are very heterogeneous, it would seem that 1,25 (OH)2D is able to activate NK cells and, consequently, offers the potential for use in cancer patients subjected to treatment with immune checkpoint inhibitors.

 

Vitamin D and Cardiovascular Diseases

Vit D receptors are widely expressed in the human body, including in cancer cells and the liver, kidneys, and cardiovascular system. Recently, it was confirmed that VDRs are expressed both in vascular endothelial cells and in cardiomyocytes, modulating their mitochondrial metabolism, calcium homeostasis, and the production of endocardial metalloproteases.

 

A recent publication found that low serum levels of 25OHD are associated with a high risk of cardiovascular diseases, including stroke, heart failure, and general cardiovascular mortality. A recent meta-analysis confirmed, however, that Vit D supplementation does not reduce adverse cardiac events, either in elderly or in non-elderly patients. In addition, it was demonstrated that exogenous Vit D supplementation does not reduce cardiovascular mortality, heart attack, MACE, or myocardial infarction.

 

However, it is assumed that Vit D supplementation in pediatric patients may reduce cardiovascular mortality in adulthood, possibly due to epigenetic modifications that improve cardiovascular health.

 

Furthermore, observational studies confirmed, within their secondary objectives, the existence of an inverse association between serum Vit D levels and the incidence of heart diseases, although several confounding factors may alter the interpretation of the data, and a conclusion cannot be unequivocally determined.

 

The primary outcomes were Vit D supplementation and the risk of osteoporosis in postmenopausal women and the reduction in the risk of dialysis in subjects with renal insufficiency, respectively. It is therefore important to emphasize that these studies always involved older people, or, in any case, people not of premenopausal age; therefore, the effective long-term benefit of Vit D for cardiac events was not included as a primary outcome.

 

Consequently, further studies of cardiovascular outcomes should be designed, using both a younger population and higher doses of Vit D. Another recent study confirmed that the early administration of high-dose enteral Vit D provided no benefit over placebo in terms of mortality or other nonfatal outcomes in critically ill patients with Vit D deficiency. By contrast, other studies indicate that daily Vit D supplementation at 2000 IU for three months improves endothelial function and reduces the expression of oxLDL and ICAM1 (cardiovascular risk factors) in patients with hypertension and type 2 diabetes. Another, the similar, study showed that the daily administration of 2000 IU and 800 IU of Vit D lowered mean systolic blood pressure over an observation period of two years.

 

 

Vitamin D and Human Microbiota

The human microbiota is the community of commensal, symbiotic, and pathogenic microorganisms that a human harbor. It consists of about 10–100 trillion microbial cells of bacteria, archaea, fungi, algae, and small protists. The collection of genes harboring these microorganisms forms the human microbiome.

 

The most diverse, abundant, and well-studied human microbiota is that of the gut, but the microbiota of the skin, lung, oral cavity and genitourinary tract are also proving to be important features in human pathophysiology. Due to the emerging importance of the microbiota for human health and behavior, the human microbiota is now even considered an additional “organ”.

 

The gut microbiota comprises four main phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, the first two of which are found in abundance in human feces. Genetic and non-genetic factors influence the composition of the microbiota, resulting in a dynamic environment. However, diet is responsible for most of the changes in the composition of the gut microbiota. Several pieces of evidence indicate that the human microbiota affects the production and degradation of nutrients and human metabolites, such as Vit D and its active metabolites.

 

Indeed, Vit D has been shown to both affect and be affected by the gut microbiota: the latter altered intestinal Vit D metabolism, while probiotic supplements affected circulating 1,25(OH)2Dlevels. Moreover, Vit D has been linked to a wide range of biological activities, including the modulation of gut mucosal immunity and the integrity of the intestinal barrier and mucus layer, and it is mediated by VDR.

 

VDR is highly expressed in normal gut epithelial cells, especially in the crypts. The Vit D /VDR signaling pathway plays a major role in maintaining gut homeostasis through the regulation of tight junctions and adherent junction components, as well as the release of antimicrobial peptides, such as defensins.

 

 

Vitamin D and Overall Risk of Infection

Vit D also deeply influences the immune system, by acting directly on immune cells to promote an anti-inflammatory state; Vit D deficiency encourages the development of a proinflammatory state. Accordingly, there is solid evidence that Vit D supplementation can reduce the rate of infection. For example, it has been shown that Vit D status can impact the development and severity of acute respiratory tract infections, such as influenza and pneumonia.

 

A recent meta-analysis determined that Vit D3 supplementation significantly decreases the risk of developing acute respiratory tract infections: supplementation was especially effective in participants who presented low serum levels of 25OHD at baseline.

 

Vitamin D has also been associated with disease severity in COVID-19 patients. Those who presented Vit D deficiency on admission had a greater risk of both requiring invasive mechanical ventilation and mortality. Patients with COVID-19 were found to have a higher prevalence of Vit D deficiency than control groups. Vit D has recently been demonstrated to increase the expression of ACE2, the main receptor of SARS-CoV-2, which facilitates SARS-CoV2 host-cell invasion in vitro.

 

Higher concentrations of this receptor can also promote a greater pro-inflammatory response, which may exacerbate organ damage in patients. Indeed, one small Italian study established a possible increased risk of in-hospital mortality due to Vit D supplementation.

 

 

Conclusions

We conducted a systematic review of both observational and interventional studies to comprehensively evaluate the effects of Vit D intake on chronic diseases, particularly cardiovascular disease and cancer. Vit D is a fat-soluble vitamin that acts as a steroid hormone, and its primary source is the UVB-induced conversion of 7-dehydrocholesterol to Vit D in the skin. Secondary sources include Vit D-containing foods and dietary supplements.

 

Regardless of the source, Vit D undergoes multiple hydroxylation reactions to its active form, 1,25-dihydroxy vitamin D, which can exert biological effects. Vit D activity is recognized as part of the endocrine system, maintaining extracellular calcium levels via the regulation of both intestinal calcium absorption and osteocyte turnover. Most of the biological activities of this steroid hormone are exerted by binding to the vitamin D receptor.

 

VDR is one of the nuclear receptors for steroid hormones that functions as a ligand-activated transcription factor, thereby regulating gene expression.

 

Although VDR is expressed primarily in the small intestine, bones, and kidneys, organs that are sensitive to vitamin D because of its central role in calcium homeostasis, this receptor has also been found in other tissues and organs, including the skin, and some cell types in the immune system, suggesting a possible influence of Vit D on the immune response to various diseases. It is well known that childhood rickets is a direct consequence of severe vitamin D deficiency in the diet.

 

However, the association between vitamin D deficiency and osteomalacia in adults is more controversial. Daily supplementation with 400 IU of vitamin D may prevent rickets and osteomalacia when serum 25OHD levels rise above 30 nmol/L, which is considered the threshold for vitamin D deficiency. In any case, supplementation with Vit D alone seems not to be the solution for reducing fracture risk. A combination of calcium and vitamin D supplementation (1000 mg and 800 UI daily respectively) can reduce the incidence of hip and other fractures in adults by 20% by both increasing 25OHD serum levels by 25 nmol/L and improving BMD and bone quality.

 

Muscle homeostasis is also related to Vit D, which affects muscle development and strength. Muscle weakness is typical in individuals with chronic renal dysfunction due to the depletion of 1,25(OH)2D and in individuals with genetic alterations in CYP27B1 and VDR. Vit D deficiency is also associated with an increased risk of infections and immune and autoimmune diseases. In particular, there is a strong association between low serum levels of 25OHD and the risk of developing multiple sclerosis.

 

Considering the role of 1,25(OH)2D in the downregulation of the adaptive immune system, it is conceivable that Vit D deficiency promotes autoimmune diseases, such as multiple sclerosis, inflammatory bowel disease, and type 1 diabetes. Current knowledge of vitamin D status and cardiovascular disease risk indicates that there is no clear benefit of vitamin D supplementation in individuals at risk of heart failure, myocardial infarction, and stroke. However, few data are available to draw a conclusion, and specific studies are needed to better elucidate the role of vitamin D supplementation in individuals with cardiovascular disease.

 

Data support the association between low serum Vit D levels and type 2 diabetes mellitus and metabolic syndrome, especially the association between the slowing of the progression from prediabetes to T2DM and Vit D supplementation. Recently, the role of the microbiota in human health has become increasingly important, and several studies are underway that aim to better understand the relationship between the composition of the gut microbiota and the development of disease in children and the elderly.

 

Studies both in vivo and in humans support the benefits of Vit D supplementation for gut microbiota composition, although some questions remain about the proper 25OHD serum level to improve the microbiota–Vit-D axis. In the context of the COVID-19 pandemic, the relationship between vitamin D status and the severity of SARS-CoV disease has received attention.

 

Observational studies and meta-analyses associated low serum Vit D levels with both high COVID-19 mortality and morbidity, with many confounding factors, including a similarity between the risk factors for Vit D deficiency and for COVID-19. However, recent studies suggest that Vit D supplementation is of no benefit in hospitalized patients with COVID-19 and that there is no association between genetic serum 25OHD levels and the risk of hospitalization in COVID-19 patients. Thus, it is difficult to draw a definitive conclusion about the role of Vit D in reducing the risk of severe SARS-CoV-2 infection.

 

There are strong preclinical and epidemiological studies linking Vit D to cancer risk, proliferation, and prognosis.

 

The role of Vit D in carcinogenesis appears to be multifactorial, suggesting a possible role in the control of cellular processes, such as inflammation, proliferation, angiogenesis, differentiation, invasion, and apoptosis. Altogether, epidemiologic studies are still inconclusive in determining the true effect of vitamin D on reducing cancer risk and improving patient outcomes. New, adequately designed RCTs must be performed.

 

However, it can be said that supplementation with Vit D and its analogs is a promising strategy for both the prevention and treatment of cancer. Moreover, the use/support of Vit D in a particular set of patients, such as those affected by HIV infection and cancer, should be mandatory, with the aim of reducing the risk of opportunistic infections, mainly during anticancer treatment. For patients, an adequate serum value of Vit D should reduce the risk of cancer and immunity deficiency.

 

Most importantly, a recent meta-analysis revealed that the oral administration of Vit D reduces plasma levels of MDA, a marker of lipid peroxidation, at doses of 100,000 and 200,000 IU per month. The antioxidant mechanisms of Vit D are related to its membrane and lipoprotein scavenger activities, which can reduce iron damage, ferroptosis, and ROS production.

 

 

 

 

 

 

 

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Story Source:

Berretta, M.; Quagliariello, V.; Bignucolo, A.; Facchini, S.; Maurea, N.; Di Francia, R.; Fiorica, F.; Sharifi, S.; Bressan, S.; Richter, S.N.; et al. The Multiple Effects of Vitamin D against Chronic Diseases: From Reduction of Lipid Peroxidation to Updated Evidence from Clinical Studies. Antioxidants 2022, 11, 1090. [https://doi.org/10.3390/ antiox11061090] Published: 30 May 2022