Clinical Significance of Analysis of Vitamin D Status in Various Diseases

Vitamin D plays a role not only in the proper functioning of the skeletal system and the calcium-phosphate equilibrium, but also in the immune system, the cardiovascular system and the growth and division of cells. Although numerous studies have reported on the analysis of vitamin D status in various groups of patients, the clinical significance of measurements of vitamin D forms and metabolites remains ambiguous. This article reviews the reports analyzing the status of vitamin D in various chronic states. Particular attention is given to factors affecting measurement of vitamin D forms and metabolites. Relevant papers published during recent years were identified by an extensive PubMed search using appropriate keywords. Measurement of vitamin D status proved to be a useful tool in diagnosis and progression of metabolic syndrome, neurological disorders and cancer. High performance liquid chromatography coupled with tandem mass spectrometry has become the preferred method for analyzing the various forms and metabolites of vitamin D in biological fluids. Factors influencing vitamin D concentration, including socio-demographic and biochemical factors as well as the genetic polymorphism of the vitamin D receptor, along with vitamin D transporters and enzymes participating in vitamin D metabolism should be considered as potential confounders of the interpretation of plasma total 25(OH)D concentrations.


Introduction
Vitamin D involves six different steroid hormones with different activities: the inactive endogenous precursor-cholecalciferol (D 3 ), partially active calcidiol (25(OH)D 3 ), its active dihydroxy-form calcitriol (1,25(OH) 2 D 3 ), a plant-derived inactive form-ergocalciferol (D 2 ), and its metabolites, 25(OH)D 2 and 1,25(OH) 2 D 2 [1]. Calcitriol has a key role to play in calcium and phosphate homeostasis, cell proliferation and differentiation, and in the responses of the immune and nervous systems [2]. In the last two decades novel functions of vitamin D have been discovered including protection against cardiovascular disease, diabetes and colorectal cancer, neuroprotective activity, oxidative stress reduction, xenobiotic detoxification, and antimicrobial and anti-inflammatory activity [3]. Vitamin D's mechanism involves binding calcitriol to the vitamin D receptor (VDR), which is a transcription factor positively or negatively regulating the expression of genes that mediate its biological activity. Wide distribution of the VDR in all cell types may explain its multiple actions on different tissues [4]. As the function of VDR may be influenced by circulating levels of vitamin D, assessment of vitamin D status in the body may be clinically valuable to predict 25(OH)D and 1,25(OH)2D can be converted by CYP24A1 to 24,25(OH)2D and 1,24,25(OH)3D, respectively. 24,25(OH)2D appears to be required for optimal endochondral bone formation and bone fraction repair [20]. It possesses a circulating half-life of seven days and concentrations in ng/mL (0.084-9.514 ng/mL [16]). The metabolite is regarded as a useful marker of vitamin D catabolism [21]. In patients with CYP24A1 mutations, 24,25(OH)2D levels are low or even undetectable despite the presence of adequate amounts of the substrate 25(OH)D. Low concentration of 24,25(OH)2D and normal-to-high concentrations of 25(OH)D have been found in children with idiopathic infantile hypercalcemia carrying mutations of CYP24A1 [22]. It was suggested that 1,24,25(OH)3D may have biological activity because it shows substantial affinity for the VDR [2]. The enzyme can also catalyze the C23 lactone pathway resulting in the formation of 1,25(OH)2D-26,23 lactone and 25(OH)D-26,23 lactone [4]. Thus, the primary function of CYP24A1 is to prevent the accumulation of 1,25(OH) 2 D and 25(OH)D. Moreover, vitamin D metabolites may undergo an epimerization process by 3epimerase. The enzyme isomerizes the C-3 hydroxy group of the A ring from the α to β orientation. Clinical significance of the 3-epimers is unknown but reduced binding of 3-epi-25(OH)D to DBP and reduced affinity of 3-epi-1,25(OH) 2 D for the VDR were reported [2]. Evidence suggests that 3-epi-1,25(OH)2D suppresses PTH as effectively as 1,25(OH)2D. Moreover, 3-epi-1,25(OH)2D3 can boost the synthesis of surfactant phospholipids and affect gene expression to activate the synthesis of surfactant protein-B in pulmonary alveolar type II [23]. Levels of the 3-epimers in the human body are low and rarely detected. Serum concentrations of 3-epi-25(OH)D3 in normal subjects were in the range of 0.4-6.0 ng/mL [16]. In keratinocytes, 20-hydroxylation of vitamin D by CYP11A1 takes place, resulting in formation of 20(OH)D and 20,23(OH) 2 D. Biological activity of 20(OH)D3 was confirmed in the induction of keratinocyte differentiation [24]. It was reported that the compound inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells through activation of VDR [25]. 25(OH)D and 1,25(OH) 2 D can be converted by CYP24A1 to 24,25(OH) 2 D and 1,24,25(OH) 3 D, respectively. 24,25(OH) 2 D appears to be required for optimal endochondral bone formation and bone fraction repair [20]. It possesses a circulating half-life of seven days and concentrations in ng/mL (0.084-9.514 ng/mL [16]). The metabolite is regarded as a useful marker of vitamin D catabolism [21]. In patients with CYP24A1 mutations, 24,25(OH) 2 D levels are low or even undetectable despite the presence of adequate amounts of the substrate 25(OH)D. Low concentration of 24,25(OH) 2 D and normal-to-high concentrations of 25(OH)D have been found in children with idiopathic infantile hypercalcemia carrying mutations of CYP24A1 [22]. It was suggested that 1,24,25(OH) 3 D may have biological activity because it shows substantial affinity for the VDR [2]. The enzyme can also catalyze the C23 lactone pathway resulting in the formation of 1,25(OH) 2 D-26,23 lactone and 25(OH)D-26,23 lactone [4]. Thus, the primary function of CYP24A1 is to prevent the accumulation of 1,25(OH)2D and 25(OH)D. Moreover, vitamin D metabolites may undergo an epimerization process by 3-epimerase. The enzyme isomerizes the C-3 hydroxy group of the A ring from the α to β orientation. Clinical significance of the 3-epimers is unknown but reduced binding of 3-epi-25(OH)D to DBP and reduced affinity of 3-epi-1,25(OH)2D for the VDR were reported [2]. Evidence suggests that 3-epi-1,25(OH) 2 D suppresses PTH as effectively as 1,25(OH) 2 D. Moreover, 3-epi-1,25(OH) 2 D 3 can boost the synthesis of surfactant phospholipids and affect gene expression to activate the synthesis of surfactant protein-B in pulmonary alveolar type II [23]. Levels of the 3-epimers in the human body are low and rarely detected. Serum concentrations of 3-epi-25(OH)D 3 in normal subjects were in the range of 0.4-6.0 ng/mL [16]. In keratinocytes, 20-hydroxylation of vitamin D by CYP11A1 takes place, resulting in formation of 20(OH)D and 20,23(OH)2D. Biological activity of 20(OH)D 3 was confirmed in the induction of keratinocyte differentiation [24]. It was reported that the compound inhibits DNA synthesis in epidermal keratinocytes, melanocytes, and melanoma cells through activation of VDR [25].

Mechanism of Action
1,25(OH) 2 D 3 , the active form of vitamin D, acts as steroid hormone by binding to VDR that is omnipresent throughout the body. VDR is a transcription factor that forms a complex with another intracellular receptor, the retinoid-X receptor (RXR). The genomic mechanism of vitamin D action involves the direct binding of 1,25(OH) 2 D 3 activated VDR/RXR to specific DNA sequences called vitamin D response elements (VDREs) resulting in either activation or inhibition of transcription. By turning genes on or off, this complex controls intestine calcium and phosphate absorption, renal calcium reabsorption and phosphate loss, and bone homeostasis. Other VDR dependent effects involve regulation of hormone secretion, cell differentiation and proliferation, and immune function. 1,25(OH) 2 D inhibits PTH production and secretion by PTH gene suppression, stimulates insulin secretion in pancreatic beta cells, and stimulates fibroblast growth factor (FGF) production in osteoblasts and osteocytes. It was reported that vitamin D slows down cancer cell growth by such mechanisms as stimulation of the expression of cell cycle inhibitors p21 and p27, activation of the expression of the cell adhesion molecule E-cadherin, inhibition of the transcriptional activity of b-catenin, and controlling angiogenesis [26]. The immunomodulatory effect of 1,25(OH) 2 D 3 involves stimulation of antimicrobial peptides, cathelicidin and defensin β2, reducing T cell proliferation, modulating T cell differentiation, and inhibition of the maturation of dendritic cells important for antigen presentation [27].
1,25(OH) 2 D also has effects on selected cells that do not involve gene regulation and are mediated by a membrane receptor. This mechanism regulates calcium and chloride channel activity, activation and distribution of protein kinase C, and phospholipase C activity in osteoblasts, liver, muscle, and intestine cells [2].

Methods of Analysis of Vitamin D in Biological Fluids
Serum levels of the biologically active metabolite 1,25(OH) 2 D may be difficult to measure because half-life of this compound is about 4 h and its production is regulated by hormones and minerals [28]. 25(OH)D is employed as a biomarker for overall vitamin D status because its half-life is two-three weeks and its concentration accounts for both dietary vitamin D intake and endogenous synthesis of vitamin D in the skin [14]. Moreover, levels of the main circulating form, 25(OH)D 3 , have been correlated to the onset and progression of many diseases [29,30]. Therefore, precise and accurate methods for analysis of 25(OH)D are essential to distinguish insufficient levels of the compound.
Vitamin D and its metabolites are demanding analytes to determine, because they are very lipophilic, at very low concentrations, and also have a high affinity for proteins that bind to vitamins [1]. There are several FDA-approved commercial 25(OH)D tests for routine clinical laboratory measuring of vitamin D status based on radioimmunoassay (RIA), enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), and competitive protein-binding assay (CPBA) techniques [6].

Immunoassay Techniques
A large number of immunoassay methods for measurement of the vitamin D forms exist. They differ in antibodies used and as a result some of them measure only one form, 25(OH)D 2 or 25(OH)D 3 , while others measure total 25(OH)D levels [31]. Recently, a rapid immunoassay-based point-of-need diagnostic test for the assessment of 25(OH)D 3 in finger-stick blood was developed. The assay was accompanied by a smartphone-assisted imaging device which allows for easy operation and access to the results [32]. Moreover, a chemiluminescent immunoassay method was developed for determination of the sum of 1,25(OH) 2 D 2 and 1,25(OH) 2 D 3 . However, the obtained results exhibited significant deviation from the values determined by a reference HPLC-MS/MS method [33]. It was reported that immunoassay methods showed significant discrepancies in results, demonstrating positive bias, which results in overestimating vitamin D deficiency [34]. Moreover, Lee et al. [35] suggest that difference in cross-reactivities may be the main cause of discrepancy between immunoassays.

HPLC-MS/MS
Recently, HPLC-MS/MS has come to represent the gold standard for vitamin D plasma evaluation because it can discriminate between all forms and metabolites of vitamin D. Moreover, MS/MS detection allows the analysis of much lower concentrations of vitamin D and its metabolites than previously used HPLC-UV methods which enabled the determination of 25(OH)D 2 and 25(OH)D 3 only. Recently, interest in the other forms and metabolites of vitamin D has arisen. The measurement of 25(OH)D using standard chromatographic methods may result in overestimated values because of the presence of the C-3 epimers, which may constitute up to 50% of the 25(OH)D content in adults [36,37]. Clinically, this issue is particularly important in estimating vitamin D status in infants where levels of the epimeric form may be higher than concentrations of 25(OH)D [38].
There are several HPLC-MS/MS methods for analysis of vitamin D in human serum, plasma, dry blood spots and milk [16,19,[39][40][41][42][43][44][45][46][47][48][49][50][51][52]. Moreover, application of supercritical fluid chromatography (SFC) with MS/MS detection was reported [13,53]. These methods allow the analysis of not only the main metabolite 25(OH)D but also substrates, D 2 and D 3 , as well as other metabolites including epimers and dihydroxyl derivatives ( Table 1). In the methods described, the chromatographic separation of vitamin D and its metabolites was usually performed on a C18 column. For more efficient separation of multiple isomeric compounds, a chiral column with cellulose selector was also used [16,53]. The mobile phases were composed of organic reagents such as methanol or acetonitrile with the addition of an aqueous solution of formic acid, ammonium formate, ammonium acetate or methylamine. For SFC, mixtures of CO 2 with methanol and formic acid were used (Table 1).
In these methods, an effective release of 25(OH)D from the binding protein can be assured by a simple sample preparation procedure including protein precipitation or alkaline hydrolysis followed by liquid-liquid extraction (LLE) with hexane or ethyl acetate or solid-phase extraction (SPE). In one case, immunoextraction was used to purify the sample [19]. The various extraction methods have enabled effective isolation of analytes from biological material, as evidenced by the recovery value in the range of 50-113% [48,49]. Some procedures required further derivatization with 4-(4 -dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) or 4-phenyl-1,2,4-triazoline-3,5-dione were (PTAD) to improve sensitivity of the method in case of 1α,25(OH) 2 D 3 [53] or to generate a stable product ion under MS ionization for 25(OH)D 3 glucuronide [48]

Analysis of Vitamin D in Various Diseases
In the case of vitamin D deficiency in the body, it was proved that the parathyroid glands produce too much PTH, which then contributes to an increase in resorption in bone tissue metabolites, leading to a decrease in mineral density, which leads to a general feeling of tiredness and weakness, and as a consequence osteomalacia and osteoporosis [54]. Therefore, vitamin D deficiency may be associated with an increased risk of fracture [55].
Vitamin D deficiency very often occurs in patients with autoimmune diseases such as rheumatoid arthritis (RA), lupus erythematosus, or a number of other rheumatological conditions [56]. Therefore, it seems that in these conditions measurements of vitamin D levels may be very important ( Table 2). Serum concentrations of metabolites such as 3-epi-25(OH)D 3 and synovial fluid metabolites, especially 1,25(OH) 2 D 3 , were found to be more closely related to the progression and course of RA [55]. Li et al. [57] reported that 3-epi-25(OH)D 3 was significantly lower in serum of RA patients and in resolving reactive arthritis (ReA) patients relative to healthy controls. Moreover, the authors found differences in concentrations of various vitamin D metabolites between serum and synovial fluid [57]. In RA, active forms of vitamin D, in response to current inflammation, reduce the number of Th1 and Th17 lymphocytes and reduce the production of pro-inflammatory cytokines, which are responsible for severe RA symptoms [58]. In another study [59], the effects of vitamin D supplementation in RA patients have contributed to improving patient health. Disease activity expressed by the mean disease activity index using C-reactive protein in these patients showed statistically significant improvement after supplementation (p = 0.002). Serum vitamin D levels increased from 10.05 ± 5.18 to 57.21 ± 24.77 ng/mL (p < 0.001) during treatment [59].
Vitamin D deficiency has been associated with autoimmune thyroid disease (Hashimoto's, Graves' disease) and non-inflammatory bowel disease. Serum 25(OH)D levels were significantly lower in patients with hypothyroidism compared to the control group (t = −11.11; p = 0.000). As a result of poor absorption caused by inflammation of the intestines, the deficiency of vitamin D is worsened due to impaired food absorption [60]. Another example of the effects of vitamin D deficiency is psoriasis-a chronic inflammatory disease that increases the risk of cardiovascular disease. A proportional relationship was demonstrated between 1,25(OH) 2 D and risk factors for cardiovascular disease, such as the volume of visceral adipose tissue (β = −0.43, p = 0.026 and β = −0.26 p = 0.13), uptake of fluorodeoxyglucose (FDG) in vessels (β = −0.19, p = 0.01) and coronary plaque load (β = −0.18, p = 0.03), regardless of traditional risk factors. It can be concluded that the level of 1,25(OH) 2 D helps to better capture the cardiometabolic risk associated with vitamin D deficiency [61].
Evidence suggests that insulin resistance syndrome (metabolic syndrome) and vitamin D deficiency contribute greatly to the formation of cancer [62]. High insulin levels may cause obesity, hypertension, low HDL cholesterol, high triglycerides, and diabetes [63]. It is interesting that vitamin D deficiency in the body contributes to the deepening of insulin resistance, resulting in metabolic disorders (hyperglycemia, dyslipidemia) [64].
The presence of a vitamin D receptor in endothelial cells, smooth blood vessels and cardiomyocytes and the presence of 1α-hydroxylase in the heart confirm the effect of this compound on the circulatory system. Regulation of renin-angiotensin-aldosterone (RAA), and modulation of the inflammatory response and tissue calcification process are involved in the pathogenesis of cardiovascular diseases, which include myocardial infarction, atrial fibrillation, or heart failure [65]. As a result of vitamin D deficiency, an increase in the concentration of parathyroid hormone is observed, which leads to the development of hypertension, endothelium dysfunction and calcification of the aortic valve. Calcitriol, by reducing the level of free radicals, has anti-inflammatory effects, thereby affecting the stabilization of atherosclerotic plaques [66].
Among the many positive properties of 1,25(OH) 2 D, the anti-cancer effect should also be taken into account. This compound works by inhibiting proliferation, activating apoptosis, stimulating differentiation, and inhibiting angiogenesis [67]. Studies have shown a directly proportional relationship between the incidence of given types of cancers and the concentration of vitamin D in serum and latitude. People with vitamin levels higher than 50 mmol/L had a lower risk of developing prostate and colorectal cancer by as much as 30-50% [12,68]. While a link exists between vitamin D levels of <30 nmol/L in the blood and increased incidence of colorectal cancer (31%), in the case of breast and prostate cancer no associations were found [69,70]. In the study of Manson et al., after one year of supplementation with 2000 IU of vitamin D, average levels of 25-hydroxyvitamin D increased by 40%. However, vitamin D supplementation did not result in a lower incidence of invasive cancer [71].
Vitamin D deficiency has also been noted in patients with chronic obstructive pulmonary disease (COPD), obesity, high triglycerides, diabetes and its complications, metabolic syndrome, and multiple sclerosis. For diabetes, measuring 1,25(OH) 2 D 3 serum may help to predict the severity of diabetic retinopathy [72]. In people with metabolic syndrome, low vitamin D levels were more common in people with a higher BMI [73,74]. Low levels of 25(OH)D in the blood may contribute to a higher risk of developing multiple sclerosis in newborns. This risk of developing the disease was the highest at 25(OH)D levels <20.7 nmol/L, while at ≥48.9 nmol/L, risk was lowest [75].
Another inflammatory disease in which vitamin D pathogenesis can be important is bronchiolitis in infants. This may suggest a negative correlation between low concentrations of 25(OH)D and IgE in the serum of small patients [76]. Infants who suffered from food allergies were also diagnosed with vitamin D deficiencies that increased the likelihood of persistent allergies (especially in people with the GG genotype in vitamin D-binding protein). Antenatal supplementation has been shown to reduce the risk of food allergies in particular in infants with GT/TT genotype [77].
Low serum vitamin D levels may be associated with the onset of psychiatric disorders such as depression and post-traumatic stress disorder (PTSD) [78][79][80]. It was found that vitamin D is involved in the processes of maturation and differentiation of the cells of the nervous system, by affecting the synthesis of neural and glial cell lines of growth factors. It also controls the synthesis of the neurotransmitters acetylcholine (ACh), dopamine (DA) and γ-aminobutyric acid (GABA) [81]. Changed expression of the vitamin D receptor gene has been shown to be associated with Alzheimer's disease. Vitamin D may have neuroprotective properties, protecting against the harmful effects of glutamate and reducing inflammation induced by the deposition of β-amyloid in the form of plaques [81]. Interesting conclusions are provided by the study of Jhee et al. [78] regarding the occurrence of depression in people with chronic kidney disease. It was noted that vitamin D deficiency was an important independent predictor of depression, and the incidence was significantly higher in patients with chronic kidney disease than in the general population (14.3 vs. 11.1%, p = 0.03). In addition, the study by Imai et al. [79] showed that stronger symptoms of depression occurred in those with the lowest levels of vitamin D. Due to vitamin D deficiency, anatomical changes in the hippocampus have been observed and serotonin levels and calcium levels in neurons have been reduced, which also predisposes to depression [82]. Insufficient skin synthesis of vitamin D in the autumn-winter period leads to a higher probability of the appearance of depressive states, which disappear in the summer [83]. Terock et al. [80] observed that deficient 25(OH)D levels were positively associated with post-traumatic stress disorders (OR = 2.02; p = 0.028). Additionally, in carriers of the CC-genotype of rs4588, significantly higher 25(OH)D levels were observed (p < 0.001) along with lower odds for the disease (OR = 0.35; p = 0.023) compared to the AA-genotype. Carriers of the TT-allele of rs7041 showed lower 25(OH)D levels (p < 0.001) and increased odds for the disease (OR = 2.80; p = 0.015) compared to the GG-genotype [80].  Association of vitamin D deficiency (<50 nmol/L) with: • reduced % predicted forced expiratory volume in one second (p for trend = 0.06) • reduced % predicted forced vital capacity (p for trend = 0.003).
[77]  [80] * not specified if plasma or serum was used for 25(OH)D measurements. HOMA-IR-homeostasis model assessment of insulin resistance.
Vitamin D deficiency is a risk factor for acute respiratory infections (ARI). Meta-analysis by Martineau et al. [84] confirmed that vitamin D supplementation protects against ARI. Recently, several studies have started to clarify whether supplementation with vitamin D in different dosages has an influence on the course of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [85,86].

Factors Influencing Vitamin D Status in Human Body
Vitamin D regulates calcium and phosphorus metabolism [87]. The compound plays an antagonistic position against the parathyroid hormone and stimulates PTH secretion by inducing calcium removal from bones. The level of vitamin D, and more precisely its active metabolite 1α, 25(OH) 2 D 3 , is under the control of PTH and depends on the content of calcium and phosphorus in the body [17]. Epidemiological studies suggest that even mild vitamin D deficiency increases PTH levels and may worsen bone condition. An increase in the value of calcidiol in serum from 20 to 32 ng/mL (50-80 nmol/L) improves calcium transport efficiency in the intestine by 45-65% [88]. In addition, calcitriol is regulated by fibroblast growth factor (FGF-23) produced by bone tissue. High blood calcitriol levels induce expression of the enzyme D-24-hydroxylase (CYP24A1), which may transform calcitriol into the inactive compound calcitroic acid [89]. This is a feedback system that regulates the concentration of active vitamin metabolic agents and prevents the body from hypervitaminosis D [22]. The relationship between PTH and low vitamin D level may be an important determinant mainly in the case of older people. With a decrease in vitamin D levels, calcium and phosphorus absorption becomes suboptimal. PTH level and alkaline phosphatase activity rise [90]. In people with low levels of 25(OH)D, and increase in PTH concentration, ionized calcium level may also be higher. This suggests that in similar populations vitamin D levels <25 ng/mL can be recognized as a cut-off point for starting vitamin D supplementation [91]. Alvarez et al. confirmed that 25(OH)D blood concentrations are independently associated with plasma glutathione, thiol redox status systems and inflammatory markers [92]. The metabolism of vitamin D also depends on magnesium, which acts as a cofactor for 25-hydroxylase, 1α-hydroxylase, and 24-hydroxylase. Moreover, the activity of the vitamin D binding protein (VDBP) is magnesium dependent [93]. Therefore, a low level of this mineral may change the link between 25(OH)D in serum and PTH [94]. Vitamin D anti-inflammatory properties were manifested by inhibiting the kappa B (NFκB) nuclear pathway. Serum 25(OH)D concentrations were positively associated with NFκB activity in peripheral blood mononuclear cells [95].
Vitamin D 3 synthesis and its conversion into 25(OH)D 3 depend on various socio-demographic and biochemical factors ( Figure 2).
Among environmental factors, latitude, season, and residence significantly impact D 3 skin synthesis. The status of vitamin D in the body also varies depending on ethnicity, gender, and age ( Table 3).
The chances of vitamin D deficiency increase with age and lack of sun exposure. Vitamin D synthesis is most effective in the summer between 10 am and 3 pm in light-skinned people living below 40 degrees latitude [96]. In areas below the 34th parallel north, the amount of solar energy is large enough for vitamin D synthesis to occur throughout the whole year. Still, these regions' inhabitants also have deficiencies, especially women living in the Middle East, who avoid the sun for religious reasons and cover the whole body, including the face [97,98]. Therefore, a low level of this mineral may change the link between 25(OH)D in serum and PTH [94]. Vitamin D anti-inflammatory properties were manifested by inhibiting the kappa B (NFκB) nuclear pathway. Serum 25(OH)D concentrations were positively associated with NFκB activity in peripheral blood mononuclear cells [95].
Vitamin D3 synthesis and its conversion into 25(OH)D3 depend on various socio-demographic and biochemical factors (Figure 2). Among environmental factors, latitude, season, and residence significantly impact D3 skin synthesis. The status of vitamin D in the body also varies depending on ethnicity, gender, and age (Table 3). There are several reports of the effect of residence on vitamin D levels. Griffin et al. [99], stated that different lifestyle led by rural residents, compared to urban residents, could be associated with a risk of vitamin D deficiency. The authors explained that rural dwellers are less exposed to sunshine because of changes in lifestyle as they now commute longer distances to work in the cities. Additionally, older rural dwellers may be less active due to geographic isolation and have less access to information on the benefits of good vitamin D health. It was shown that 25(OH)D levels in serum were significantly lower among rural residents in western Ireland, regardless of age. The deficiency of this vitamin was lower for women both in the city and in the countryside [99]. Moreover, Carpenter et al. [100] reported that concentrations of 25(OH)D depend on ethnicity, and are positively correlated with European ancestry (p < 0.001), and negatively correlated with African ancestry (p = 0.016).
Other natural factors that regulate vitamin D synthesis in the skin are skin pigmentation, sunscreen creams (SPF), and clothing. Large amounts of melanin in dark skin effectively block vitamin D synthesis. Melanin reduces the epidermis' penetration by UVB, limiting the dermal synthesis of 25(OH)D [101]. Therefore, people of color require more prolonged exposure to the sun than Caucasians [102,103]. Sunscreen use with sun protection factor (SPF) 15 reduces cholecalciferol synthesis by up to 99% [104]. Most current health messages suggest several short sun exposure sessions in the summer to ensure sufficient vitamin D production. The guidelines indicate that 25% of the skin's exposure for 15-30 min between 10 am and 3 pm, two or three times a week, may meet the demand for vitamin D in Caucasian people [96]. The evidence of Touvier et al. [105] also suggests that even low daily-life sun exposure behaviors contribute to increased vitamin D synthesis and improved 25(OH)D concentrations. Unfortunately, this does not apply to everyone. The amount that can be obtained in this way varies considerably from person to person and is influenced by several factors such as high BMI, race, ethnicity and genetic factors, namely polymorphisms in key genes of the vitamin D pathway, modulating vitamin D status [106]. However, during a study by Joh et al. [102], both sun exposure (≥20-30 min/day around noon) and 500 IU vitamin D 3 supplementation significantly affected serum 25(OH)D levels compared to placebo. Supplementation alone, compared to UV exposure, caused a more significant increase in vitamin levels.
Deficiency of vitamin D is primarily a common health problem for the elderly. It is associated with decreased physical performance, and a higher risk of cognitive impairment, depression and anxiety. In people over 70, the amount of vitamin D precursor 7-dehydrocholesterol may be reduced by up to 60-75% [107]. Older people are vulnerable to many risk factors, such as reduced dietary intake, reduced exposure to sunlight (living in nursing homes), increased skin thickness, impaired intestinal absorption, and impaired hydroxylation process in liver and kidney. Older women had significantly lower levels of this vitamin [108]. However, Vitamin D deficiency occurs at all ages, mainly in children from 12 to 14 years old [109]. Numerous studies indicate a negative correlation of vitamin D concentration with parameters such as BMI, percentage of body fat, or WHR among children. It was proven that the rate of vitamin D deficiency in girls was higher than among boys. The level of this vitamin in the body could be influenced by adolescents' behaviour and eating habits. It was shown that 25(OH)D levels correlated with obesity factors, mainly in boys. It is believed that girls have proportionally more subcutaneous fat, which poses a lower health risk than visceral and abdominal fat. Estrogens may also play a significant role in the metabolism of vitamin D in girls [109].
Obesity correlates with vitamin D deficiency through reduced bioavailability of this compound due to lower physical activity and lower sun exposure [110,111]. According to Monache et al., 25(OH)D concentration in serum correlated with BMI, regardless of season and age. Lower vitamin D values characterized women with obesity compared to women with healthy body weight and overweight in winter and summer [112,113]. Therefore, the developed guidelines recommend a twice-daily dose for obese people than the recommended dose for peers with appropriate body weight [114]. Low levels of vitamin D in the body may be caused by reduced bioavailability from the gastrointestinal tract due to impaired absorption or inability to use stored fat deposits in obese people. Another probable mechanism of decreased vitamin D concentration in this group is the increased synthesis of the active metabolite of vitamin D-1,25(OH) 2 D 3 in the kidneys. This metabolite inhibits the production of 25(OH)D 3 in the liver in a negative feedback mechanism [89].
Vitamin D deficiency correlates with diet. Cases of reduced vitamin D levels have been found in vegetarians, vegans, and macrobiotic nutritionists [115]. Interestingly, supplementation with high doses of vitamin D contributes to a change in the human intestinal microbiome. A decrease in the Bacteroidetes and Lactobacillus bacteria and an increase in Firmicutes and Bifidobacterium were observed [116].
Smoking may also be a predisposing factor for lowering vitamin D levels (<20 ng/mL). There is a hypothesis that smoking interferes with the conversion of 25(OH)D to 1,25(OH) 2 D, thus reducing calcium absorption in the gastrointestinal tract, leading to loss of bone mineral density, especially in men over 65 years of age [91,117]. Some medicines, e.g., antiepileptic drugs (phenytoin, phenobarbital), rifampicin, orlistat (used to treat obesity), cholestyramine, glucocorticosteroids, and immunosuppressants can reduce vitamin D levels [106].
DBP is a multifunctional protein in an ascetic fluid, plasma, the cerebrospinal fluid also found on the surface of numerous cells. It binds to the different forms of vitamin D including ergocalciferol, cholecalciferol, calcifediol, and active calcitriol [118]. Different physiological and pathological conditions can affect DBP levels, which in turn affect circulating concentrations of vitamin D forms. Carpenter et al. proved that 25(OH)D levels are positively correlated with circulating DBP (R = 0.25, p < 0.001) [100]. Moreover, a significant number of children (52.7%) with vitamin D deficiency had low DBP < 168 mg/L (p = 0.015) [119]. In pregnancy and liver diseases, the affinity of DBP for vitamin D metabolites may be decreased. It was reported that DBP level increases two-fold between the second and third trimesters while mean free 25(OH)D lowers and total 25(OH)D remains unchanged [120]. Liver diseases result in reductions in DBP. In patients with liver cirrhosis levels of free 25(OH)D were higher than in healthy subjects [121]. Therefore, alterations in DBP levels should be considered as potential confounders of the interpretation of plasma total 25(OH)D concentrations. Bikle and Schwartz [122] suggest that assessment of vitamin D status might be improved by measuring free 25(OH)D instead of, or in addition to, total 25(OH)D.
Gene mutations are important factors affecting the synthesis and metabolism of vitamin D. They include polymorphisms of CYP450 isoenzymes, protein transporters, and VDR. In the case of impaired catabolism of 25(OH)D and 1,25(OH) 2 D caused by mutations in the CYP24A1 gene encoding 24-hydroxylase, or the fact of excessive synthesis of 1,25(OH) 2 D resulting indirectly from the mutation in the SLC34A1 gene encoding the sodium co-transporter phosphate (NaPi-IIA) in the kidney, elevated calcitriol concentration is observed [123]. The risk of hypervitaminosis D in both cases is increased with the use of prophylactic doses of vitamin D. The polymorphism of the gene responsible for the metabolic pathway of vitamin D (GC gene-rs2282679) encodes DBP, which is responsible for regulating the transport of 25(OH)D and 1,25(OH) 2 D to tissues. Studies in various populations have shown a strong association between GC-sr2282679 and vitamin D status at lower 25(OH)D levels in people with the CC genotype, suggesting the effect of vitamin D on length telomeres. The fact that people with CC genotype had lower LTL (leukocyte telomere length) than people with AA genotype confirms the hypothesis regarding the long-term effect of vitamin D on telomere shortening [124]. However, in many bigger population studies, no relationship was found between 25(OH)D serum concentrations and LTL [125]. The VDR gene is located on the long arm of chromosome 12 (12q12-14) and has about 200 single nucleotide polymorphisms (SNPs). The Bsm variant of the VDR gene was associated with hypertriglyceridemia and may be predisposed to metabolic syndrome (MetS). The VDR TaqI TT and BsmI BB + Bb genotypes were associated with lower 25(OH)D levels in the metabolic syndrome group [126]. Moreover, in obese patients with vitamin D deficiency, carriers of polymorphic alleles showed significant lower levels of serum 25(OH)D and higher HOMA-IR (the homeostasis model assessment of insulin resistance; p = 0,04), blood pressure levels (p < 0.001) and lipid parameters compared to those with the wild type homozygotes (p = 0.02) [127]. Cdx2 polymorphism may be a potential biomarker in vitamin D treatment for breast cancer, irrespective of VDR receptor expression. The B allele or Bb VDR genotype presence may be a risk factor for breast cancer development [128].
Several studies proved that after supplementation with 25(OH)D 3 , vitamin D serum levels appeared to be genetically modified [118,[129][130][131][132]. Carriers of the common AA genotype of rs4588 polymorphism better respond to vitamin D supplementation [118]. People carrying allele G of CYP2R1 had a higher vitamin D concentration after nine weeks of supplementation [131]. Moreover, CYP2R1 and CYP24A1 DNA methylation levels could potentially be biomarkers of variability in vitamin D responses. People with high levels of DNA methylation in two genes may need high vitamin D supplementation to achieve optimal 25(OH)D serum levels [132]. Table 3. Factors influencing vitamin D status in human body.

Factors
Studied Group (N) Conclusions Ref.
Significant correlations between Ca 2+ and PTH (r = −0.223, p < 0.001), 25(OH)D and PTH (r = −0.178, p < 0.001) and between PTH and age (r = 0.322, p < 0.001) were found. [17] calcium, PTH, alkaline phosphatase 58 children and adolescents A positive and significant correlation was found between dietary calcium and vitamin D (r = 0.77, p < 0.001). [90] sun exposure (<30 min and ≥30 min per week) PTH ionized calcium 1339 ≥18 years old The median of 25(OH)D <10 ng/mL associated with hypercalcemia. The levels of 25(OH)D were higher in women who received >30 min of sun exposure per week, and who claimed to use sunscreen <3 times/week (p ≤ 0.001). [91] glutathione and cysteine thiol/di-sulfide redox status 693 adults (449 females, 244 males) Serum 25(OH)D was positively associated with plasma GSH and negatively associated with plasma redox potentials-Eh GSSG and Cys (p < 0.001 for all). [94] nuclear factor kappa-B activity 49 In healthy adults, 25(OH)D concentrations were positively associated with NFκB activity in peripheral blood mononuclear cells (r = 0.48, p = 0.0008). [95] place of residence: urban area, rural area, season, sex 17,590 (urban n = 4824; rural n = 12,766) Serum 25(OH)D concentrations were lower among rural compared to urban dwellers and depend on sex (p < 0.001) and age (for urban p < 0.001, for rural p < 0.001). [99] Ancestry, vitamin D binding protein 750 healthy children [100] vitamin D supplementation, sun exposure (>20 min/day during summer, and >30 min/day during fall) 50  Vitamin D status was lower among women (p < 0.0001), older subjects (p = 0.04), obese or underweight subjects (p < 0.0001), subjects who lived at higher latitudes (p < 0.0001), and those whose blood draw occurred in early spring (p < 0.0001). Vitamin D status was higher among subjects who were more physically active (p < 0.0001), who had higher scores of usual sun exposure (p < 0.0001), those with higher Fitzpatrick photo-type (p = 0.03).

Factors Studied Group (N) Conclusions
Ref.
nutritional and lifestyle patterns 116 Vitamin D deficiency was associated with higher systolic ambulatory and daytime blood pressure monitoring (p = 0.01 and p = 0.02, respectively), lower step counts, lower urinary calcium, and higher fat mass. Milk intake (p = 0.009) and fish (p < 0.001) were lower in the deficient. [113] vitamin D supplementation, microbiome 50 adolescent girls before and after vitamin D supplementation The mean (±SD) of serum vitamin D level at baseline was 11 ± 9 ng/mL and after high dose vitamin D supplementation it increases to 40 ± 17 ng/mL (p < 0.001). A high dose supplementation of vitamin D alter the human gut microbiome composition: Bacteroidetes and Lactobacillus fell by 72% and 24% respectively, whilst Firmicutes and Bifidobacterium were increased by1. 5  Levels of free 25(OH)D were higher in patients with cirrhosis (p < 0.0033) while DBP concentrations were lower than in other groups and differed between whites and blacks (p < 0.0033) and between DBP haplotypes (p < 0.0001). [121] rs12785878, rs10741657, rs6013897, rs2282679 461 (33-79 years old) Participants with CC genotype (rs2282679) had shorter age-and sex-adjusted mean leukocyte telomere length (LTL) than those with AC and AA genotypes (p < 0.05). Serum 25(OH)D concentrations were not associated with LTL. [124] FokI BsmI TaqI Cdx2 237 participants with metabolic syndrome (MetS), 376 controls VDR TaqI TT, and BsmI BB + Bb genotypes were associated with lower 25(OH)D levels (p < 0.05) in comparison to TaqI Tt, and BsmI bb genotypes in the MetS group. Cdx2 GG genotype was associated with higher waist circumference compared with the AG genotype in all subjects (p < 0.05). BB + Bb genotypes of the VDR BsmI had significantly increased the odds ratio of hypertriglyceridemia when compared with the bb genotype (OR 1.87, p = 0.022).
[126] In obese with vitamin D deficiency, carriers of polymorphic alleles showed significant lower levels of serum 25(OH)D and higher HOMA-IR (the homeostasis model assessment of insulin resistance; p = 0.04), blood pressure levels (p < 0.001) and lipid parameters compared to those with the wild type homozygotes (p = 0.02). [127] receptor gene BsmI (A/G) polymorphism 60 females with breast cancer (BC) 25(OH) vitamin D levels were significantly lower in the patients with BC compared to controls (p ≤ 0.001). Carriers of Bb genotype had 4.6 times increased risk of developing breast cancer when compared to other genotypes. [128] BsmI polymorphism of the VDR gene, supplementation

elderly women with vitamin D insufficiency
Supplementation with a vitamin D 3 megadose reduced inflammatory markers and increased the total antioxidant capacity in elderly women with vitamin D insufficiency (p = 0.03). The 25(OH)D (p = 0.0001), PTH (p = 0.009), us-CRP (p = 0.007) and α1-acid glycoprotein (p = 0.005) levels of elderly patients with the BB/Bb genotype were more responsive to supplementation compared with those with the bb genotype.
[129] CYP2R1 27 children with rickets, 50 unrelated subjects After supplementation with 50,000 IU of vitamin D 2 or vitamin D 3 , heterozygous subjects for the L99P and K242N mutations had lower increases in serum 25(OH)D than control subjects. [130] rs4588, supplementation 619 healthy adolescent girls Polymorphism of rs4588 was associated with serum 25(OH)D both at baseline (p = 0.03) and after supplementation (p = 0.008). The subjects with common AA genotype were a better responder to vitamin D supplementation than GG. [118] CYP2R1 (rs10766197), supplementation 253 healthy girls Subjects who had homozygous major allele GG showed two-fold higher response in serum 25(OH)D than carriers of the uncommon allele A (OR = 2.1, p = 0.03). [131] DNA methylation levels of CYP2R1, CYP24A1, CYP27A1, CYP27B1 446 women supplemented with calcium and vitamin D For CYP2R1, baseline DNA methylation levels at eight CpG sites were negatively associated with the 12-month increase in serum 25(OH)D (p < 0.05). For CYP24A1, baseline DNA methylation levels at −342C and −293C were negatively associated with vitamin D response variation (p = 0.011, p = 0.025, respectively). [132] Nutrients 2020, 12, 2788 23 of 30

Conclusions
With the discovery of vitamin D-binding receptors in various cells and tissues, it was proved that vitamin D affects the proper functioning of many systems in the body including the immune system, the cardiovascular system, vision, the growth and division of cells and the formation of blood vessels. Numerous studies have confirmed the relationship of low concentrations of vitamin D and the occurrence of many diseases including rheumatoid arthritis, metabolic disorders, cancer and depression. Clinical interest in the physiological importance of vitamin D forms and their possible roles in pathophysiological processes requires an appropriate method for their measurement. It is increasingly apparent that laboratories using chromatographic based methods for 25(OH)D analysis, should separate the epimeric form of 25(OH)D 3 in order to provide completer and more accurate 25(OH)D results. Therefore, identification of a rapid, simple, reliable, and cost-effective method for the determination of vitamin D status could help in monitoring disease progression and therapeutic strategies, improving quality of care. When analyzing vitamin D status, various socio-demographic, biochemical and genetic factors influencing vitamin D synthesis and metabolism should be taken into account. Funding: The Authors would like to thank Poznan University of Medical Sciences for financial support of the paper.

Conflicts of Interest:
The authors declare no conflict of interest.