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Introduction

Vitamin D is a fat-soluble vitamin that is stored in the liver and fatty tissues of the body. Perhaps best known for its role in maintaining calcium balance and bone health, vitamin D plays critical roles in many physiological processes, such as blood pressure regulation, immune function, and cell growth. Poor vitamin D status is implicated in the pathogenesis of many acute and chronic diseases, including rickets, osteoporosis, multiple sclerosis, and cancer. Recent evidence suggests that vitamin D deficiency is associated with poor outcomes in COVID-19.[1]

Vitamin D is a controversial topic in the scientific community and has contributed to much debate regarding terminology, testing, optimal blood concentrations, repletion strategies (including dose, frequency, and vitamer), and outcomes.

This article provides an overview of a wide range of topics related to vitamin D, including its biosynthesis, recommended intakes, mechanisms, and physiological actions.

Biosynthesis of vitamin D is a multistep process.

Unlike other vitamins – trace nutrients that must be consumed in the diet – vitamin D is a steroid hormone that is produced in the body. Its synthesis occurs in a stepwise manner that requires exposure to ultraviolet B light and heat.[2] Equally important in the regulation of vitamin D in the body is a sixth enzyme, 24-hydroxylase (CYP24A1), which catalyzes the degradation of vitamin D.[3] All of these enzymes are magnesium dependent, and evidence suggests that magnesium deficiency impairs vitamin D metabolism.[4] [5]

Multiple vitamers of vitamin D exist

Five forms (referred to as vitamers) of vitamin D have been identified, but the two primary vitamers relevant to human health are ergocalciferol, commonly referred to as vitamin D2, and cholecalciferol, commonly referred to as vitamin D3.

Vitamin D2 is produced by invertebrates, some plants, and fungi. It is particularly abundant in mushrooms following exposure to ultraviolet B light.[Source: USDA)] Vitamin D3 is produced by animals, including humans, and is the vitamer typically added to foods (in fortified products) and used in over-the-counter dietary supplements. Prescription vitamin D supplements in the United States have historically provided D2 [6], but D3 is now available in prescription form.

Vitamin D vitamers are derived from a variety of sources

Vitamins D2 and D3 can be obtained from dietary sources, such as mushrooms, salmon, and many fortified foods, or from supplemental sources. The two vitamers, whether in dietary or supplemental form, are taken up in the gut and incorporated into chylomicrons (small fat globules composed of protein and lipids), pass into the lymphatic system, and then enter the blood. They are biologically inert and must be converted to the intermediate form, 25(OH)D, and subsequently to the final form, 1,25(OH)2D; however, the intermediate product differs based on the vitamer precursor, with D2 providing 25(OH)D2 and D3 providing 25(OH)D3.

Bioequivalence of vitamins D2 and D3

Controversy exists regarding the bioequivalence of D2 and D3.[6] A 2008 double-blind, randomized study involving 68 healthy adults between the ages of 18 and 84 years provided participants a placebo, 1,000 international units, or IU, of either D2 or D3, or a combination containing 500 IU of D2 and D3 daily for 11 weeks. At the end of the study period, the authors of the study noted no differences in the participants' vitamin D levels and concluded that the two vitamers were equally effective in maintaining vitamin D status.[7]

However, more recent evidence has emerged suggesting that D2 exerts lower bioactivity than D3. A 2012 meta-analysis of seven studies concluded that D3 was more effective than D2 at raising serum concentrations of vitamin D, possibly due to differences in aspects of the two vitamers' metabolism. Frequency of administration played a role in the response to supplementation. However, the authors of the analysis conceded that several factors might influence these findings, including age, sex, and ethnicity of study participants.[8]

A 2012 double-blind, randomized study involved 107 adults who took 2,000 IU of either D2 or D3 or a placebo daily for eight weeks. The authors of the study noted that supplementation with D3 increased total concentrations of 25(OH)D more efficiently than D2. Interestingly, supplementation with D2 was associated with a decrease in 25(OH)D3.[9] Similar results were found in 2018 when a randomized, cross-over trial involving 12 healthy young men demonstrated that D2 was roughly half as effective as D3 in maintaining serum concentrations of vitamin D.[10]

Vitamin D transport is targeted

Approximately 90 percent of the 25(OH)D3 in serum is bound to the vitamin D binding protein (also known as the group-specific component), a highly conserved protein that transports vitamin D and its metabolites to target tissues throughout the body.[11] The binding protein participates in numerous physiological processes, ranging from transporting vitamin D2 and D3 metabolites and binding fatty acids to possible roles in inflammation and immune function.[12] Vitamin D binding protein exhibits a strong affinity for 25(OH)D3 but a weak affinity for 1,25(OH)2D. This reduced affinity for the active steroid hormone is essential for the vitamin's activity, because binding to the protein inhibits the steroid's accessibility to the cell and prolongs 1,25(OH)2D half-life.[13]

Multiple factors influence attachment to the vitamin D binding protein

Variation in the genes that encode for the vitamin D binding protein influence 25(OH)D levels. For example, single-nucleotide polymorphisms modulate vitamin D binding protein levels and subsequent affinity for 25(OH)D3.[14] [15] Interestingly, serum concentrations of the binding protein do not appear to influence binding capacity.[16] Similar to the enzymes involved in vitamin D metabolism, concentrations of vitamin D binding protein are magnesium dependent.[5]

Vitamin D deficiency

Approximately 40 percent of people living in the United States are vitamin D deficient (as defined by the Endocrine Society, see "Vitamin D status controversy," below).[17] Deficiency is associated with osteomalacia in adults (referred to as rickets in children). Certain populations are at greater risk for deficiency, especially dark-skinned people, such as those of African or Hispanic descent, and those who are obese, have low education, smoke, or have diabetes.[17]

Osteomalacia is a skeletal disorder characterized by soft, improperly formed bones, growth retardation, muscle weakness and spasms, low blood calcium, and seizures. Rickets, which was commonly associated with children living in the industrialized cities of northern Europe and the northeastern United States during the 18th and 19th centuries, has re-emerged as a public health problem in recent decades in parts of Southeast Asia as well as in exclusively breastfed infants in the United States.[18] [19] [20]

The increased risks for vitamin D deficiency are likely due to both modifiable and non-modifiable factors. For example, exposure to ultraviolet B light is the primary source of vitamin D, but behaviors or characteristics that block or reduce sun exposure, such as using sunscreen, having high levels of melanin (a dark pigment found in skin that acts like natural sunscreen), exclusive breastfeeding, older age, or being obese can impair the skin’s ability to produce vitamin D3.

Interestingly, findings from a recent analysis suggest that the risks associated with sunscreen use are low; however, much of the research was based on use of sunscreens with low protective factors, rather than newer generation sunscreens that have higher protective factors.[21] Similarly, dark skin impairs vitamin D synthesis but to a very low extent and might not fully explain why vitamin D status is often poor in people with dark skin.[22]

Exclusively breastfed infants are at risk of deficiency because vitamin D concentrations are low in breast milk, even if maternal intake is considered adequate.[23] However, epidemiological studies indicate that women living in northern latitudes who breastfeed are more likely to be vitamin D deficient, further increasing the risk of deficiency in their infants.[24] A randomized controlled trial demonstrated that maternal supplementation of 6400 IU during breastfeeding is sufficient to meet infant needs.[25] In lieu of maternal supplementation, the American Academy of Pediatrics recommends that all infants have a minimum daily intake of 400 IU of vitamin D beginning soon after birth to reduce the risk of deficiency.[26]

Older age can also have a profound effect on the skin's ability to produce vitamin D3. The skin of a person in their seventies produces roughly 25 percent of the vitamin D3 produced by a person in their twenties.[27] Nearly two-thirds of older adults living in the United States are vitamin D deficient.[28]

The obesity epidemic also plays a role in the prevalence of vitamin D deficiency. Vitamin D is a fat-soluble vitamin that is readily stored in adipose tissue, which acts as a "sink" for vitamin D precursors and intermediate forms, preventing conversion into the active hormone form. In addition, the activity of enzymes that participate in vitamin D synthesis is impaired in obese people.[29]

Other factors contributing to vitamin D deficiency include conditions that impair intestinal absorption of vitamin D, such as celiac disease or gastric bypass. In addition, people who have chronic liver disease or kidney disease are at high risk for vitamin D deficiency because the enzymatic hydroxylation of the vitamin D precursors occurs in the liver and kidneys. Furthermore, many medications accelerate vitamin D inactivation, including commonly prescribed blood pressure medications such as nifedipine and spironolactone, as well as clotrimazole (an antibiotic), and rifampin (an anti-tuberculosis drug), among others.[30]

Vitamin D synthesis begins in the skin with exposure to sunlight and heat and continues in the liver and the kidneys. Vitamin D3 obtained in the diet or via supplements is taken up in the gut and eventually enters the bloodstream. Once there, it undergoes the same transformative processes in the liver and kidneys as sun-derived vitamin D3.

The Food and Nutrition Board of the National Academies of Science (formerly the Institute of Medicine) has established recommended dietary allowances, or RDAs, for daily vitamin D (as D2 or D3) intake for all ages and life stages of people living in the United States. These RDAs have been deemed sufficient to prevent nutritional rickets and osteomalacia and maintain normal calcium metabolism in healthy people. They were established based on presumed minimal sun exposure.

The RDAs for vitamin D are commonly presented in both International Units (IU) and micrograms (mcg), with a biological activity ratio of 40 IU to 1 microgram.

RDAs vary according to age and life stage. Amounts are typically listed in both micrograms (mcg) and international units (IU); 1 mcg of vitamin D is equal to 40 IU.

Upper intake levels vary according to age and life stage. Amounts are typically listed in both micrograms (mcg) and international units (IU) and include vitamin D from all external sources (foods, beverages, and supplements).

Tolerable upper intake levels of dietary and supplemental vitamin D intake vary across the lifespan and life stage. They range from 1,000 IU per day for infants to 4,000 IU per day for people aged 9 years and older to reduce the risk of kidney and tissue damage associated with hypercalcemia.[31]

Challenges to the current RDAs

A substantial number of experts from multiple disciplines has called for a review of the current dietary recommendations for vitamin D, arguing that the recommendations are far too low to maintain optimal health.[32] As described above, the RDAs for vitamin D were established based on the vitamin's effects on calcium homeostasis, not other important physiological roles, such as immune function. In recent decades, emerging data indicate that vitamin D status influences risk for many chronic diseases, such as metabolic syndrome and it related disorders, cardiovascular diseases, autoimmune disorders, cancer, and many other causes of premature death.[33] The effect of vitamin D supplementation remains controversial and the need for intensive study is great. The many physiological roles of vitamin D will be discussed in detail in later sections of this article.

Assessment of vitamin D status

The circulating half-life of 25(OH)D3 is approximately 15 days.[34] Consequently, its concentration in serum is the most reliable indicator of vitamin D status, reflecting not only endogenous synthesis of the vitamin but also dietary and supplemental intake.[35] However, serum levels of 25(OH)D3 levels do not indicate the amount of vitamin D stored in body tissues. Parathyroid concentrations are regarded as a functional measure of vitamin D status.[36]

Serum levels of 25(OH)D3 are typically reported in both nanomoles per liter (nmol/L) and nanograms per milliliter (ng/mL). Some evidence suggests that adverse effects can occur when serum levels are greater than 125 nmol/L (50 ng/mL), especially when levels exceed 150 nmol/L (60 ng/mL). Toxicity can occur when serum 25(OH)D3 levels exceed 250 nmol/L (100 ng/mL), but these levels are likely achievable only through continuous oral intake greater than 10,000 IU (250 micrograms) daily.[37] Symptoms of toxicity involve multiple organ systems, including neurological, cardiovascular, renal, and gastrointestinal systems. The most common symptoms include confusion, apathy, recurrent vomiting, abdominal pain, frequent urination, excessive thirst, and dehydration.[38]

Vitamin D assays

Further complicating the assessment of vitamin D status is the variability associated with the assays used to measure serum 25(OH)D3 concentrations, which can provide falsely low or high results.[39] These assays are generally categorized as binding assays or chemical assays.

Binding assays include chemiluminescence immunoassay, radioimmunoassay, and binding protein assay. The specificity of the antibodies used and the extensive binding of vitamin D to its transporter protein hinders the accuracy of these assays. Chemical assays include high performance liquid chromatography and liquid chromatography-mass spectrometry, or LC-MS, and require high level expertise to utilize. Each of the methods has its advantages and disadvantages, but LC-MS is widely considered the "gold standard" of vitamin D assessment.[40]

Another factor complicating the accuracy of vitamin D assessment lies in the vitamin's capacity to convert to epimers. Epimers are forms of a chemical compound that differ slightly in terms of their symmetry around the carbon molecules in their structure. Evidence suggests that all vitamin D vitamers can convert to epimers at their third carbon, commonly referred to as C3.[41] Higher levels of C3-epimers are prevalent in pregnant women and infants.[42] One review suggested that total D3 concentrations in adults contain an average of 6 percent C3-epimer.[43] Many assays do not differentiate between C3-epimers, leading to misclassification of vitamin D status. However, LC-MS/MS can distinguish C3-epimers from other forms of vitamin D.[44]

Vitamin D status controversy

In recent decades, controversy has arisen regarding the terminology used to describe vitamin D status.

In 2011, a committee of the Institute of Medicine, or IOM, concluded that serum 25(OH)D concentrations less than 30 nmol/L (less than 12 ng/mL) places people "at risk of vitamin D deficiency"; concentrations ranging from 30 to 50 nmol/L (12 to 20 ng/mL) places some populations "at risk for inadequacy"; and concentrations of 50 nmol/L (20 ng/mL) or greater are considered "sufficient."[45]

That same year, the Endocrine Society convened a task force to establish guidelines for clinicians for the evaluation, treatment, and prevention of vitamin D deficiency. The task force's findings closely mirrored those of the IOM, with some differences. They suggested that concentrations less than 20 ng/mL (50 nmol/L) define "deficiency," and concentrations ranging from 52.5 to 72.5 nmol/L (21 to 29 ng/mL) define "insufficiency."[46] They also recommended widespread testing for at-risk populations.

The IOM committee members responded to the Endocrine Society's position, acknowledging that certain populations might be at greater risk for poor vitamin D status. However, they argued that the serum concentration cutoffs the Endocrine Society established were incorrect, and the recommendations for widespread testing were unnecessary and costly. They further posited that the Endocrine Society's position was not evidence-based.[47]

Supplementation

The effectiveness of vitamin D supplementation is dose- and frequency-dependent. An analysis of data collected from more than 2,700 people revealed that consuming 1,000 to 2,000 IU of vitamin D twice a week for one month did not increase 25(OH)D levels. Higher doses were more effective, however. For example, 2,000 to 3,000 IU elicited a 7 percent increase; 3,000 to 4,000 IU elicited a 13 percent increase; and 5,000 IU elicited a 30 percent increase. Frequency influenced the efficacy of supplementation, as well. For example, taking a supplement between three and six times per week increased vitamin D levels by roughly 5 nmol/L to 16 nmol/L. However, taking a supplement seven times per week promoted a 30 nmol/L increase. A final factor in increasing vitamin D levels was duration, with supplementation that lasted five or more months eliciting an increase of more than 6 nmol/L.[48]

Vitamin D receptor and gene regulation

The active steroid hormone form of vitamin D regulates the expression of more than 900 genes in the body,[49] roughly 5 percent of the human protein-encoding genome. It accomplishes this by binding to the vitamin D receptor in the cell. This couplet binds with another receptor called the retinoid X receptor, or RXR, and then recruits various co-regulatory proteins, forming a large complex. The complex travels into the cell’s DNA where it recognizes a unique sequence – a repeat of six nucleotides separated by three spacers – called a vitamin D response element. More than 1,000 genes carry this particular sequence in their DNA. The sequence can determine whether vitamin D activates or inactivates a gene, thereby regulating the gene's function.

Vitamin D in human health

Vitamin D plays roles in calcium balance, bone health, blood pressure regulation, immune function, aging, and many other physiological processes.

Vitamin D, calcium homeostasis, and bone health

Vitamin D's principal role in maintaining calcium homeostasis is to facilitate calcium absorption in the gut. The body maintains tight control over circulating calcium levels via a multi-tissue axis and feedback loop involving calcium's intestinal absorption, bone release and uptake, and renal reclamation and excretion. Key players in these processes include parathyroid hormone, calcitonin, vitamin D, and phosphorus.

When serum calcium is low, the parathyroid glands release parathyroid hormone, which signals bone cells to release calcium and promotes reabsorption of calcium in the kidneys.[50] Parathyroid hormone also promotes the synthesis of 1,25(OH)2D3, which drives intestinal uptake of dietary calcium. The vitamin D receptor, which is expressed in all segments of the small and large intestine, mediates the mineral's absorption. It is noteworthy that dietary calcium intake is the preferred source of systemic calcium.[51] If serum calcium concentrations fall, calcitonin, a hormone produced in the thyroid gland, signals the cessation of calcium release from bone to reduce circulating blood calcium levels. Vitamin D also mediates phosphorus absorption in the gut, thereby serving as a regulator of new bone formation.[52]

Evidence from rodent and human studies indicate that aging elicits many physiological and anatomical changes in the intestine and kidneys that contribute to altered regulation of calcium homeostasis and promote age-related bone loss.[53]

In the intestine, calcium uptake decreases, promoting secondary hyperparathyroidism, which drives calcium release from bone.[54] In addition, the hydroxylation of 25(OH)D3 in the kidneys may become impaired.[55] Furthermore, the expression of CYP24A1, the enzyme that promotes the degradation of vitamin D, increases.

In the kidneys, poor overall function and reduced glomerular filtration rate occur with aging due to progressive structural deterioration of the kidney. Poor glomerular filtration correlates with decreased serum levels of 1,25(OH)2D3.[56] Interestingly, parathyroid hormone levels are elevated with age, but renal production of 1,25(OH)2D3 in response to parathyroid hormone decreases.[57] In addition, renal vitamin D receptor number decreases with age.[58]

Vitamin D participates in many aspects of bone health, including the aforementioned participation in calcium homeostasis. The most common form of age-related bone loss is osteoporosis, a condition characterized by low bone mass and structural deterioration of bone tissue. Osteoporosis promotes bone fragility and increases the risk of hip, spine, and wrist fractures. More than 53 million people living in the United States have osteoporosis or are at high risk of developing the condition.[59] Interestingly, the evidence from randomized controlled trials investigating the efficacy of vitamin D improving bone health and reducing falls has been mixed.

Synergistic effects of vitamin D and vitamin K

Some evidence points to a synergistic effect between the actions of vitamins K1 and K2 (collectively referred to as vitamin K) and vitamin D.

Vitamin K is a fat-soluble vitamin. The body has limited vitamin K storage capacity, so the body recycles it in a vitamin K redox cycle and reuses it multiple times. Naturally occurring forms of vitamin K include phylloquinone (vitamin K1) and a family of molecules called menaquinones (vitamin K2). Vitamin K1 is synthesized by plants and is the major form in the diet. Vitamin K2 molecules are synthesized by the gut microbiota and found in fermented foods and some animal products (especially liver). Vitamin K plays critical roles in blood clotting, bone metabolism, prevention of blood vessel mineralization, and regulation of various cellular functions.

Vitamin D promotes the production of several proteins that must undergo the chemical process of carboxylation to function properly. Carboxylation is a vitamin K-dependent process, and these proteins are aptly named vitamin K-dependent proteins. Scientists have identified 17 vitamin K-dependent proteins, many of which participate in regulation of soft-tissue calcification. The two primary proteins involved in this regulation are osteocalcin and matrix Gla protein. Osteocalcin is produced in the bones and promotes calcium accretion in the bones and teeth[60] Matrix Gla protein is produced in the smooth muscle cells of blood vessels and plays critical roles in inhibiting vascular calcification, a process associated with the development of cardiovascular disease and renal dysfunction.[61]

Evidence indicates that between 10 and 40 percent of the key proteins involved in regulation of soft-tissue calcification are not fully carboxylated in healthy adults, suggesting a degree of insufficiency exists in the general population.[62] High vitamin D intake may promote the production of vitamin K-dependent proteins, driving the demand for carboxylation and decreasing the body's vitamin K pool.

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