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Relationship between vitamin D deficiency and psychophysiological variables: a systematic review of the literature

Vitamin D is a fat-soluble vitamin that plays a role not only in calcium homeostasis, but also in several other functions, including cell growth and immune functions, and is considered a neurosteroid. Vitamin D deficiency is highly prevalent worldwide and has been suggested to be associated with an increased risk of emotional disorders. Therefore, the association between vitamin D levels and psychophysiological disorders, such as depression, anxiety, and mood, has been investigated. To list these variables, a bibliographical literature research was conducted in the MEDLINE/PubMed, Web of Science, Scopus, Science Direct and PsycINFO databases, between November and December 2020, with no year limits of publication. The studies involved humans aged between 18 and 59 years without associated diseases. This review presents evidence of the main variables involved in this association, main tools used to verify these variables, and methods used to verify circulating vitamin D levels in populations. Most studies have indicated that the main psychophysiological variables involved with vitamin D levels are depression and anxiety followed by mood, and an association has been observed between increased serum vitamin D levels and reduction in symptoms of depression, anxiety, and mood, and there is a heterogeneity of methods for assessing vitamin D. More studies are clearly needed to improve our understanding of their role in modulating the psychophysiological aspects of vitamin D levels.


' INTRODUCTION
Vitamin D is a liposoluble micronutrient acquired mainly endogenously by exposure of the skin to the sun, specifically ultraviolet B (UVB) rays (at wavelengths of 290-315 nm), which represents 80-90% of this acquisition. Its exogenous form is found in foods that naturally contain this vitamin, such as milk and its derivatives, as well as fatty fish (1)(2)(3)(4).
Skin exposure to UVB rays forms pre-vitamin D3, the skin precursor 7-dehydrocholesterol, which is present in all layers of human skin, mainly in the epidermis (4,5). Vitamins D2 and D3 are transported by vitamin D binding protein (VDBP) to the liver, where they undergo hydroxylation by vitamin D-25-hydroxylase (CYP2R1) to produce 25-hydroxyvitamin D (25 (OH) D). This is the main circulating metabolite of vitamin D, which is used to assess the individual status of vitamin D. Then, 25 (OH) D reaches the kidney, where it undergoes an additional hydroxylation by 25 (OH) D-1a-hydroxylase (CYP27B1) to 1,25-dihydroxyvitamin D (1.25 (OH) 2D or calcium), resulting in the bioactive form of vitamin D (5).
After hydroxylation in the kidney, calcitriol has the ability to regulate calcium-phosphorus balance and to stimulate the absorption of calcium and phosphorus by enterocytes. Similarly, calcitriol acts with parathyroid hormone, which stimulates calcium absorption by osteoclasts. In the kidney, the conversion of vitamin D to its hormonal form occurs, linked to calcium homeostasis (4). Similarly, it has been proposed that endothelial brain cells can convert inactive cholecalciferol into 25 (OH) D3. This can be metabolized into 1,25 (OH)2 D3 by cells, such as microglia or neurons, before being transferred to astrocytes, where it can promote binding to the vitamin D receptor and initiate gene transcription or, when in excess, is inactivated (6).
Authors have been committed to describing the physiological role of vitamin D receptors in various tissues and organs of the body, including the central nervous system (CNS) (7)(8)(9)(10). In the last two decades, variability in vitamin D levels has been used as a predictor of brain health (11)(12)(13). In this sense, its properties are not only related to calcium homeostasis, since there is a relationship with cognition and emotions, state of stress, anxiety, depression, poor sleep quality, mood, and neuropsychophysiological implications in general (14)(15)(16). It is important to note that the reduction in serum vitamin D levels, known as hypovitaminosis D, has been found in several populations, such as children, adolescents, adults, and the elderly, without distinction of race, ethnicity, or country (15,(17)(18)(19). This characteristic has been associated with different clinical conditions, including vascular, infectious diseases, osteoporosis, and certain types of cancer, as well as being intrinsically related to brain health (16,20,21).
Recently, researchers have investigated the possible association between vitamin D deficiency and the neurodegeneration process. This study was conducted with 2,716 apparently healthy participants, and it was observed that vitamin D deficiency (25 (OH) D o30 nmol/L) was associated with a reduction in brain volume, especially in the hippocampus, in addition to a lower amount of white matter when compared with subjects with sufficient vitamin D levels (X50 nmol). These results suggest that the decrease in plasma levels of circulating vitamin D may act as an indicator of brain health in this population (22).
Overall, studies have not only investigated the consequences of high levels of vitamin D in the human body. However, it is well established that levels above 100 ng/mL are considered toxic and may result in extra bone hypercalcemia, a low prevalence condition (5).
Although the literature presents studies that discuss the properties and actions of vitamin D in relation to the development and maintenance of CNS function, a systematized approach analyzes the possibilities of the relationship between the variation in circulating serum levels of this micronutrient and main related cognitive and emotional aspects. Therefore, this review aimed to evaluate the possible relationship between vitamin D levels and different psychophysiological outcomes in a healthy adult population of both sexes, as well as to establish the main tools used to measure these supposed aspects.

Search strategy
The protocol for systematic review was registered in the International Prospective Registry of Systematic Reviews (registration number: CRD42020211406).
A peer-reviewed literature search was conducted without language limitations or publication period in the Medline/ PubMed, Web of Science, Scopus, Science Direct, and Psycinfo databases. The research period without limitation per year ensured a broad overview of the literature and was considered adequate given the increasing global attention to vitamin D deficiency and psychophysiological variables. The keywords used comprised terms related to vitamin D, depression, anxiety, and psychophysiological aspects.

Inclusion and exclusion criteria
Studies of cross-sectional epidemiological projects, clinical trials, cohorts, and case studies were included if they met the following inclusion criteria: 1) studies published in all languages, without distinction of year of publication; 2) analysis of young adults aged between 18 and 59 years, avoiding the malabsorption bias caused by skin aging and of both sexes; and 3) studies that have a relationship between vitamin D deficiency and psychophysiological aspects. Animal studies that were not published in full and those with associated diseases were excluded.

Selection of studies
Two reviewers (MRMS and MLS) independently selected the titles and abstracts of the articles identified in the research. The full texts of the potentially eligible studies were obtained and evaluated later by two reviewers (MRMS and MLS). Disagreements were resolved through discussion and consultation with a third reviewer (WMAB).

Data extraction and analysis
The following data were extracted from each study by two authors of the review (MRMS and MLS): title, author, year of publication, sample, variables analyzed, main methods/ instruments of measurements, vitamin D parameters, and associated results. Discrepancies in data extraction were resolved through discussion.
Where applicable, the quality of the included studies was evaluated using the Joanna Briggs Institute Critical Assessment Tools checklist (available free of charge at https:// joannabriggs.org/critical-appraisal-tools). We evaluated the quality of studies that analyzed the relationship between vitamin D deficiency and psychophysiological variables according to the type of study. Specifically, we used the checklist for analytical cross-sectional studies (23), randomized clinical trials (24), cohort studies (25), and casecontrol studies (26) (Tables 1-4).
The overall quality of these studies was good, and the vast majority of articles met the requirements proposed by the Joanna Briggs article quality assessment medium.
Data synthesis was based on all included studies. Overcoming the range of study projects, characteristics, and variation in quality, a meta-analysis could not be performed. Instead, a narrative synthesis of the findings was considered the most appropriate way to evaluate and to report evidence.

' RESULTS
The research identified 775 records of peer reviews of the literature after removing duplicates. A total of 684 abstracts were selected, of which 28 were considered potentially relevant, as described in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses flowchart ( Figure 1). Of these, one full text of the peer-reviewed literature was not available and was not found in any online database, other was excluded after full reading because it was associated with some type of disease, and 11 were excluded after reading the full text because they did not meet the established criteria, such as age of participants. Finally, 15 studies were considered eligible and were included in this review. This systematic review investigated the main psychophysiological aspects involved in the decrease in levels of vitamin D circulating in the human body (Table 5). Among these studies, 10 were cross-sectional (27)(28)(29)(30)(31)(32)(33)(34)(35)(36).
The search found one cohort study (37), and three studies were clinical trials (38)(39)(40). One study was a case control type (41). In general, among the studies included in this review, female individuals were the most studied (35,40,41). In total, 950 women were included in the studies, and only one study conducted the research exclusively with men (33). The studies (30,32,37) did not report the sex of their population. In terms of age, all studies were of adults aged between 18 and under 60 years, according to the eligibility criteria of the study.

Vitamin D levels and the main psychophysiological aspects evaluated
Of the 15 studies included in this review, 13 investigated the association between vitamin D levels and symptoms of anxiety and depression (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Of these studies, nine were cross-sectional (28)(29)(30)(31)(32)(33)(34)(35)(36). Mood was investigated in two studies (39,40); happiness or well-being were addressed : Was true randomization used for assignment of participants to treatment groups?; Q2: Was allocation to treatment groups concealed?; Q3: Were treatment groups similar at the baseline?; Q4: Were participants blind to treatment assignment?; Q5: Were those delivering treatment blind to treatment assignment?; Q6: Were outcomes assessors blind to treatment assignment?; Q7: Were treatment groups treated identically other than the intervention of interest?; Q8: Was follow up complete and if not, were differences between groups in terms of their follow up adequately described and analyzed?; Q9: Were participants analyzed in the groups to which they were randomized?; Q10: Were outcomes measured in the same way for treatment groups?; Q11: Were outcomes measured in a reliable way?; Q12: Was appropriate statistical analysis used?; Q13: Was the trial design appropriate, and any deviations from the standard RCT design (individual randomization, parallel groups) accounted for in the conduct and analysis of the trial? Table 3 -Critical appraisal results for included studies using the Joanna Briggs Institute for verification of cohort studies.
Were the two groups similar and recruited from the same population?; Q2: Were the exposures measured similarly to assign people to both exposed and unexposed groups?; Q3: Was the exposure measured in a valid and reliable way?; Q4: Were confounding factors identified?; Q5: Were strategies to deal with confounding factors stated?; Q6: Were the groups/participants free of the outcomes at the start of the study (or at the moment of exposure)?; Q7: Were the outcomes measured in a valid and reliable way?; Q8: Was the follow up time reported and sufficient to be long enough for outcomes to occur?; Q9: Was follow up complete, and if not, were the reasons to loss to follow up described and explored?; Q10: Were strategies to address incomplete follow up utilized?; Q11: Was appropriate statistical analysis used?  in the study by Choukri et al. (40); anger, cognition, and emotion were psychophysiological aspects in the study by Dean et al. (38); and personality was investigated in the study by Avinun et al. (27).
In studies that examined the association between vitamin D levels and depressive symptoms or depression, as well as traits and/or anxiety, there was no association between low vitamin D levels and symptoms of depression or anxiety (28,38). A study by Pooyan et al. (29) showed that there was food interaction with the VDBP genotype to moderate the risk of depression; studies (30)(31)(32)34,36,41) also showed a strong association between the decrease in vitamin D levels and emergence of higher levels of anxiety and depression scores. Black et al. (37) showed a cross-sectional association between vitamin D concentrations and symptoms of depression, but not anxiety and stress. Al-Atram et al. (33) reported that vitamin D deficiency was positively correlated with anxiety symptoms, but no significant correlation (N/S) between vitamin D deficiency and depression. Lansdowne and Provost (39) evaluated vitamin D and mood levels and showed that higher levels of vitamin D3 had positive effects on mood. The study by Choukri et al. (40) analyzed an association between vitamin D deficiency, depression, and mood simultaneously in women and found that the effects of a single monthly dose of vitamin D3 supplementation during autumn and winter on depression and other mood outcomes in premenopausal healthy women were considered nonsignificant (N/S); the study evaluated pregnant women, and their results showed that low vitamin D levels at the beginning of pregnancy are associated with higher scores of depressive symptoms at the beginning and end of pregnancy (35). In addition to anxiety, Hashemi et al. (41) also observed stress and concluded that low levels of vitamin D may be associated with a higher level of stress and anxiety. Von Känel et al. (36) found that vitamin D deficiency increases the severity of depression, as well as symptoms of anhedonia.

Methods for the evaluation of circulating vitamin D
Although most methods of analysis for the evaluation of vitamin D levels used in the studies are heterogeneous, in some cases, a certain homogeneity was followed as in the studies (28,33,41) that used the enzyme-linked immunosorbent assay (ELISA) method to verify vitamin D levels; the vitamin D levels were analyzed by radioimmunoassay (29,30,35). Studies (36,37,40) used the liquid isotope dilution chromatography method for their analyses, and Black et al. (37), in addition, used liquid isotope dilution and mass spectrometry. Huang et al. (32) used liquid chromatography and mass spectrometry. Dean et al. (38) used mass spectrometry alone. Avinun et al. (27) used saliva DNA isolation using a self-collecting DNA kit. Bičíková et al. (31) performed the electrochemiluminescence immunoassay method, and Altınbas¸(34) used the electrochemiluminescence method. However, the study (39) did not report its method of analysis.

Main tools used to perform the evaluation of psychophysiological aspects
The studies (30,34,36,40) used the Hospital Anxiety and Depression Scale (HADS) as a tool for these variables. Beck's depression inventory was used in six studies (28,33,35,36,38,41), which is a widely-used tool to investigate both anxiety and depression symptoms. The Anxiety and Stress Depression Questionnaire was used in three studies (32,37,41).
(Continued) relation to reference values were reported: a study established a value of less than 40 ng/mL as insufficiency and that of greater than or equal to 40 ng/mL as vitamin D sufficiency (28). Bičíková  ' DISCUSSION Some evidence suggests that the main psychophysiological aspects related to vitamin D levels are symptoms of depression or established depression, followed by traits (obsessive-compulsive and panic disorders) and/or anxiety. These findings were observed in 13 of the 15 studies analyzed in this review. Most of the evidence is from cross-sectional studies, and the results vary considerably depending on the established parameters and tools used to test these variables.
Regarding the association between vitamin D levels and changes in brain function, rodent research has demonstrated the distribution of vitamin D receptors throughout the embryonic brain (proliferation zone), as well as in the neuroepithelium (42). These findings have optimized the relationship between vitamin D and neuromodulation aspects, which are not limited only to the brain in formation, but include the adult brain of humans and comprise several areas, such as the temporal, orbital, cingulate cortex, thalamus, accumbens nuclei, and parts of the terminal stria and amygdala, as well as pyramidal neurons of the CA1 and CA2 hippocampus regions, CA3, and CA4 (8).
Although the mechanisms involved in the pathophysiology of depression are complex and still poorly defined or established (43), the results of this study support research that establishes a relationship between the levels of vitamin D circulating in the body and depressive symptoms, because vitamin D exerts neuroprotective effects by inhibiting inflammatory cytokines, such as interleukin 6 (44). A cohort study conducted in the Netherlands between 2004 and 2007 with a population aged 18-65 years, which differs from the population aged 18-59 years established in this review, aimed to analyze the possible association between vitamin D levels and depressive symptoms; this study indicated that low levels of 25 (OH) D were associated with the presence and severity of depressive disorder, suggesting that hypovitaminosis D may represent an underlying biological vulnerability for the onset of depression. The evaluation method entailed isotope dilution-liquid chromatography of online solid phase extraction-mass spectrometry, a method that was used in some studies of this review. The tool used to assess depression severity was the self-report Inventory of Depressive Symptomatology of 28 items (45). It is noteworthy that none of the studies in this review used this tool; many of our results have also linked the effects of vitamin D to anxiety,  as this may precede depression, and both can be caused by stress (46). A recent study conducted in rats aimed to investigate the possible protective effects of vitamin D on anxiety and depression-like behaviors induced by unpredictable chronic stress and criteria for oxidative damage to brain tissue and neuroinflammation. The results do not differ from those presented in this review, since there was a positive association between vitamin D and neuropsychophysiological aspects, such as depression, anxiety, and stress. The authors also added that pre-treatment with vitamin D improved the performance of rats in the elevated cross maze, open field, and forced swimming test (47). In addition, a clinical trial conducted with older adults found no relationship between increased serum vitamin D levels and improvement of depressive symptoms, despite supplementation with increasing serum concentrations of 25 (OH) D in the intervention group to an average of 85 nmol/L after 6 months, while the placebo group remained stable at an average value of 43 nmol/L. However, the intervention had no significant effect on depressive symptoms in the study by De Koning et al. (48), which may be because of the age of the participants. Although there is no consensus on the positive performance of vitamin D and depression, some authors have found this association.
There is heterogeneity in the main laboratory methods verifying circulating vitamin D levels, as well as in our results. The choice of method is adopted not only in human studies (49) but also in animals. One study used ELISA kits both before and after vitamin D supplementation to conduct research with rats (50). Radioimmunoassays were used in three studies of this review, dating back to the 1990s and specifically used for vitamin D metabolism (51). Today, its use is well established. Studies have used this method for their research, corroborating the results found in this review (52,53). Similar to radioimmunoassays, tests with liquid chromatography were also mentioned in this review and have well-established bases to verify circulating vitamin D (54,55). Tests using mass spectrometry have been presented in recent studies and are used as a method of choice (56).
This review clarifies that the main tool used to verify depressive disorders was the Beck Inventory, which proved to be an effective measure for the identification of depressive symptoms. A study conducted with postpartum women used the Beck Depression Inventory as an observational tool for depressive symptoms (50). In addition to Beck's Inventory, the HADS has been presented as a choice to trace depressive symptoms, used in three studies. Rolf et al. (52) used this method in their study with humans; this was a clinical trial with patients with multiple sclerosis to observe if there was a difference in the scores of depression before and after vitamin D supplementation, and for this, they chose the HADS as a tool (52). The results of this review sought to identify the diversity of psychophysiological aspects involved in vitamin D mechanisms and how vitamin levels can contribute to this heterogeneity.
Although a diversity of studies addressed vitamin D levels, a consensus has not yet been well established. In our results, most studies indicate that a value of greater than 20 ng/mL is adequate to provide certain protection and/or health-related benefits in the populations studied. Most experts agree that 25 (OH) D levels of less than 20 ng/mL is considered vitamin D deficiency, while 25 (OH) D levels of 21-29 ng/mL is considered insufficient. The goal should be to keep the levels in children and adults at a level of greater than 30 ng/mL to maximize the health benefits offered by vitamin D (57)(58)(59)(60). Although some authors agree with these vitamin D levels, there is still a controversy because of the lack of consensus on the ideal range for serum 25 (OH) D (61,62).
Taken together, most experts agree that vitamin D deficiency can be defined as a 25 (OH) D level of less than 20 ng/mL, which is in agreement with the results of this review, since the severity of depressive symptoms and/or anxiety were found in patients with vitamin D levels lower than 20 ng/mL.

' CONCLUSION
This study reported some evidence regarding the main variables related to vitamin D levels and psychophysiological aspects involving the young population, highlighting the depression and/or symptoms, as well as anxiety and/or traits, followed by variation of mood status. In our research, it became clear that Beck's Depression Inventory is the most commonly used tool for investigating symptoms and depression. There is strong evidence that the increase in circulating vitamin D levels in the body plays an important role in the psychophysiophagic health of young individuals. Even lower levels of 20 ng/mL confer a greater possibility of developing psychophysiological disorders, and these higher levels are related to lower risk and/or improvement of these dysfunctions.

' AUTHOR CONTRIBUTIONS
Silva MRM conceived the review and selected titles and abstracts. Barros WMA reviewed the manuscript. Silva ML selected title and abstracts. Silva JML, Souza APS, Silva ABJ and Fernandes MSS supported the study planning. Souza SL and Souza VON guided data collection and study planning.