Next Article in Journal
Endometriotic Follicular Fluid Affects Granulosa Cells’ Morphology and Increases Duplication Rate and Connexin-43 Expression
Previous Article in Journal
Peptide-Based Regulation of TNF-α-Mediated Cytotoxicity
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biomolecules
Volume 15
Issue 4
10.3390/biom15040560
biomolecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

The Quantification of Vitamin D in Humans: A Promising, Non-Invasive and Cost-Effective Method to Measure 25-Hydroxyvitamin D

by
Giulia Squillacioti
1,†,
Samar El Sherbiny
1,†,
Veronica Lettico
1,
Federica Ghelli
1,*,
Marco Panizzolo
1,
Giacomo Scaioli
1,
Manuela Martella
1,
Selene Limoncelli
2,
Giulio Mengozzi
2,3 and
Roberto Bono
1
1
Department of Public Health and Pediatrics, University of Turin, Via Santena 5 bis, 10126 Turin, Italy
2
Clinical Biochemistry Laboratory, Città della Salute e della Scienza di Torino, Molinette Hospital, Corso Bramante 88/90, 10126 Turin, Italy
3
Department of Medical Sciences, University of Turin, Corso Dogliotti 14, 10126 Turin, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biomolecules 2025, 15(4), 560; https://doi.org/10.3390/biom15040560
Submission received: 10 March 2025 / Revised: 3 April 2025 / Accepted: 8 April 2025 / Published: 10 April 2025
(This article belongs to the Section Biological Factors)
Browse Figures
Versions Notes

Abstract

:
Background: Vitamin D intake and synthesis are essential. Vitamin D deficiency is increasing across all age groups, raising concerns regarding public health. Serum 25(OH)D is measured to define vitamin D deficiency. However, its quantification in non-invasively collected biological matrices is still poorly studied. This study aimed to assess 25(OH)D levels in unconventional matrices using cost-effective analytical methods. Methods: Serum, urine, and saliva were collected from 62 healthy, non-smoking volunteers, 25–44 years of age. Biological samples were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA). The serum was additionally analysed via the chemiluminescent microparticle immunoassay (CMIA), which was used as a benchmark. Results: We observed a linear correlation (Pearson r = 0.44; p = 0.05) between the benchmark and ELISA-measured 25(OH)D urinary levels. After stratification by sex, the correlation was stronger and significant only in females (Pearson r = 0.62; p = 0.04). Salivary 25(OH)D levels did not correlate with serum levels for both ELISA and CMIA measures. Subjects with a CMIA serum-based deficiency showed lower urinary 25(OH)D levels (p = 0.04). Conclusion: Our study opens up the possibility of using urinary 25(OH)D levels as a proxy measurement of vitamin D. Such an approach may allow future investigations on the association between environmental factors and vitamin D assessed in non-invasively collected biological matrices via cost-effective analytical methods.

Graphical Abstract

1. Introduction

Vitamin D, or calciferol, is a fat-soluble compound synthesised endogenously by human skin under sunlight irradiation (UVB rays) or obtained exogenously through the ingestion of a limited number of foods, predominantly oil-rich fish, yolks, and fortified products [1]. Vitamin D2, also called ergocalciferol, is derived from diet, while vitamin D3, or cholecalciferol, is produced through dermal synthesis when endogenous 7-dehydrocholesterol (i.e., provitamin D) is exposed to UVB rays [2]. The dermal synthesis of cholecalciferol produces approximately 80% of the total vitamin D circulating in the body. Its synthesis directly depends on the intensity of ultraviolet irradiation and indirectly on outdoor activities and environmental factors that may influence exposure to or the intensity of sunlight. Regardless of the source and form, once inside the body, vitamin D cannot exert its biological effect until it undergoes hydrolysis processes mediated by cytochrome P-450 enzymes (CYPs), including CYP2R1, CYP27A1, and CYP2D25 [3]. The first enzymatic conversion (phase I metabolism) occurs in the liver, where vitamin D is enzymatically converted into 25-hydroxyvitamin D [25(OH)D], which is the major circulating metabolite of vitamin D. Phase II metabolism involves hepatic conjugation reactions involving sulphate ester or a glucuronide moiety, which facilitate renal excretion [4,5]. Once 25(OH)D reaches the kidneys or other organs (e.g., the breasts, colon, brain, etc.), it is further hydroxylated into 1,25-dihydroxyvitamin D, the bioactive form of vitamin D [4], which circulates at much lower concentrations compared to 25(OH)D. Although the biological activity of 24,25-dihydroxyvitamin D (24,25-(OH)2D) is lower than that of 1,25-dihydroxy, it is still a bioactive form [6]. Once converted, 1,25dihydroxyvitamin D exerts its biological effects by binding to the vitamin D receptors (VDRs) expressed in various cell types, including monocytes, macrophages, dendritic cells, etc. [7]. Moreover, VDRs are highly expressed in the small intestine, where vitamin D promotes the absorption of calcium and phosphorus; in bone tissue, where vitamin D promotes osteoblast maturation to osteoclasts; and in the parathyroid glands, which release parathyroid hormone (PTH) to stimulate the renal synthesis of vitamin D in cases of low blood levels. Given its involvement in numerous biological functions, vitamin D deficiency is increasingly recognised as a widespread public health issue affecting all age groups, particularly women and the elderly [8]. Vitamin D deficiency is a major contributor to the development of metabolic bone disorders, such as hypovitaminosis predisposing to osteomalacia (rickets in children) and osteoporosis, which increase the risk of fractures. Additionally, observational epidemiological studies suggest that low vitamin D levels may be associated with the development of acute and chronic diseases, including autoimmune diseases, cardiovascular diseases, diabetes, and certain cancers [9]. Beyond non-communicable diseases, many studies have suggested that vitamin D may reduce the risk of infections, such as influenza and severe acute respiratory syndrome COVID-19 [10]. In 2020, D’Avolio and colleagues observed significantly lower levels of 25(OH)D in PCR-positive patients with coronavirus 2 (SARS-CoV-2) compared to negative patients from Switzerland, suggesting that vitamin D supplementation could play a role in preventing the risk of infection [10]. Vitamin D status is typically assessed by measuring serum- or plasma-circulating levels of the total (i.e., bound and unbound) 25(OH)D, which includes both 25(OH)D2 and 25(OH)D3 forms, with the concentration remaining stable for up to three weeks [10]. In the bloodstream, less than 1% of 25(OH)D circulates as a “free” steroid (i.e., unbounded to any proteins) [11]. Most circulating 25(OH)D is bound to a specific vitamin D binding protein (VDBP) and, to a lower extent, to albumin, both of which help regulate the total- and free-circulating levels of vitamin D metabolites [12]. Although there is ongoing debate about the optimal vitamin D level, 25(OH)D levels ranging from 50 to 125 nmol/L (20–50 ng/mL) are generally recommended. The disagreement between the Institute of Medicine (IOM), now called the National Academy of Medicine, and the Endocrine Society (ES) on optimal vitamin D levels stems from differing interpretations of evidence regarding vitamin D’s role in health. According to IOM recommendations, serum 25(OH)D levels below 20 ng/mL (50 nmol/L) are considered inadequate, while the ES considers levels below 30 ng/mL (75 nmol/L) inadequate [13,14]. In addition to the ongoing debate on optimal vitamin D levels, the lack of harmonisation in 25(OH)D quantification methods highlights the need for standardisation in this field to improve the consistency of test results, resolve discrepancies, and enhance the clinical relevance of vitamin D testing in clinical diagnosis, epidemiological studies, and research. Several analytical methods and biological matrices can be used to quantify vitamin D. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) is the gold standard for measuring vitamin D metabolites, offering high sensitivity and accuracy to simultaneously measure vitamin D metabolites such as 25(OH)D2, 25(OH)D3, and 24,25(OH)2D. However, it requires expensive equipment, long analytical times, and specialised personnel, making it less suitable for clinical laboratories. For this reason, immunological assays may be a valid alternative. Many clinical laboratories use automated immunoassays (e.g., Architect, Alinity i, Centaur, Elecsys/Modular/Cobas, Liaison, etc.) [15] and, in recent years, the ELISA (Enzyme-Linked Immunosorbent Assay) technique has gained popularity, especially in research settings [16]. Although competition between the 25(OH)D-specific antibody and VDBP may lead to poor concordance with LC-MS/MS assays [17], their relative ease of use, low cost and small sample volumes make the ELISA a promising method, particularly in research contexts. Measuring vitamin D levels through cost-effective methods in non-invasive biological matrices has become a growing area of interest for researchers and clinicians seeking alternatives to traditional blood sampling. Blood sampling for 25(OH)D concentrations is considered invasive and may cause discomfort, pain, or stress, especially in healthy individuals. Therefore, estimating 25(OH)D concentrations in non-invasively collected biological matrices, such as urine or saliva, would be highly beneficial. Although measuring 25(OH)D in saliva may not always reflect the actual levels in the body [18,19], recent studies have shown a significant positive correlation between serum and salivary vitamin D levels [20,21,22] or, less frequently, no correlation at all [23]. Moreover, since excess 25(OH)D3 is excreted in the urine following a catabolic reaction, measuring vitamin D3 in urine has also been considered potentially useful for vitamin D status determination in the body [24]. Studies have measured urine 25(OH)D levels in nephropathic patients [25], in individuals at risk of developing urinary tract infections or sepsis [26], in pregnant women [27,28], and in healthy adults [29,30]. Although promising, these approaches still require further research to overcome challenges related to sensitivity, standardisation, and correlation with blood levels. Therefore, the present study aims to investigate the feasibility of quantifying 25(OH)D levels through a cost-effective analytical method (ELISA) in non-invasively collected biological matrices (i.e., saliva and urine). Additionally, we evaluated the correlation between ELISA-based measurements of 25(OH)D in serum, urine, and saliva with a benchmark measurement of 25(OH)D assessed in serum via an automated chemiluminescent immunoassay with microparticle capture (CMIA).

2. Materials and Methods

2.1. Study Design, Setting, and Population

This study was approved by the University of Turin Bioethics Committee (Protocol No. 0654272, 24 October 2023). In September 2023, all necessary documents for obtaining approval from the Bioethics Committee were prepared. At the same time, the feasibility of the study was assessed by informally contacting potential participants among healthcare professionals in the Department of Public Health and Paediatrics at the University of Turin in Italy to gauge their willingness to participate in the study. Recruitment began on 26 October 2023, following the approval of the Bioethics Committee.
Subjects were considered eligible if they met the following criteria: if they were aged between 25 and 45 years, non-smokers, not pregnant, had taken no vitamin D supplementation within the last month, lacked any history of specific diseases (i.e., nephropathies, hepatopathies, coeliac disease, Crohn’s disease, hyperparathyroidism, psoriasis, and arthritis), and did not take antiepileptics, glucocorticoids, antiretrovirals, antimycotic, cholestyramine, or orlistat. These criteria were intended to increase the homogeneity of the sample population by reducing factors that could influence circulating vitamin D levels.
Eligible volunteers were recruited from 26 October 2023 to 3 November 2023 at the Laboratory of Environmental and Occupational Hygiene of the Department of Public Health and Paediatrics of the University of Turin. After signing the informed consent form, each participant provided fasting biological samples (i.e., blood, saliva, and urine). Procedures were performed in the early morning (7.30–9.30 a.m.). Additionally, participants were asked to complete an online questionnaire designed to gather information on their lifestyle habits and characteristics that might impact vitamin D status.

2.2. Biological Sample Collection, Processing, and Storage

Each subject provided a blood sample (8.5 mL Vacutainer® tube with separator gel, BD Franklin Lakes, NJ, USA), a drooled saliva sample (minimum 2 mL, collected in a 50 mL Falcon® Corning, Glendale, AZ, USA), and a spot urine sample (a few millilitres in a sterile 200 mL container with a screw cap). The medical personnel performing the procedures wore protective equipment and followed aseptic techniques. All biological samples were processed as detailed below and then stored at −80 °C until laboratory analysis, which was within 1 month.
To minimise risks and discomfort for participants, blood sample collection was conducted according to updated recommendations [31]. To obtain serum, whole blood was left at room temperature to clot and, within 1 h, was centrifuged at 3000 rpm for 10 min and was then aliquoted and stored. Participants provided a minimum of 2 mL of unstimulated saliva using passive drool, abstaining from tooth brushing, eating, and drinking for 30 min before sample collection. Whole saliva samples were centrifuged at 3000 rpm per 10 min, and the supernatant was aliquoted and stored. After provision, urine samples were centrifuged at 3000 rpm per 10 min, and the supernatant was aliquoted and stored.

2.3. 25(OH)D Quantification

25(OH)D levels were quantified in serum samples by an automated CMIA (Alinity i. Abbott Ireland Diagnostics Division Lisnamuck, Longford Co., Longford, Ireland), which is commonly used in the Clinical Biochemistry Laboratory (Città della Salute e della Scienza, Turin, Italy) of the Molinette Hospital to diagnose vitamin D deficiency. This measure was used as a benchmark for comparisons with the following quantifications.
Serum, saliva, and urine 25(OH)D levels were quantified using an ELISA kit and following the manufacturer’s instructions (My BioSource, MBS268910, San Diego, CA, USA). Depending on the biological matrix, specific dilution factors were applied, which were determined based on several preliminary analyses (i.e., spike-and-recovery and linearity-of-dilution experiments ). According to the Consensus Statement on Vitamin D Status Assessment and Supplementation [32], both units of measure (mol/L and ng/mL) were reported, and urine levels were corrected to the excretion rate, thus expressed as the urine creatinine ratio (ng/mg creatinine).

2.4. Other Covariates and Potential Confounders

A self-administered online questionnaire was used to collect key information on lifestyle habits and characteristics potentially impacting vitamin D status (i.e., potential confounders). In particular, adherence to the Mediterranean diet (AMD) was evaluated using the Medi-Lite standardised questionnaire, validated in 2017 [33]. The final score, from 0 (low adherence) to 18 (high adherence), was divided into tertiles describing low, medium and high AMD (the 1st, 2nd, and 3rd tertiles, respectively); details on demographics, exposure to passive smoking, anthropometric data (i.e., weight and height) and habits potentially influencing vitamin D levels (e.g., phototype, sun creams use, exposure to the sun in the last weeks, etc.) were collected using additional free questions. Self-reported weight and height were used to calculate Body Mass Index (BMI) according to the following formula: weight (kg)/height2 (metres).

2.5. Statistical Analysis

Statistical analysis was performed using the Stata (StataCorp. 2024. Stata Statistical Software: Release 18. College Station, TX, USA: StataCorp LLC.), and figures were generated using SPSS (IBM Corp. Released 2023. IBM SPSS Statistics for Windows, Version 29.0.2.0 Armonk, NY, USA: IBM Corp). Quantitative variables were summarised using the mean and standard deviation (SD) or, if skewed, the median and interquartile range (IQR), while qualitative variables were presented using absolute frequency values (number, n) and relative frequencies (%). Between-group differences in 25(OH)D levels were tested using a t-test for normally and homoscedastic distributed variables. Otherwise, we opted for one of the following non-parametric tests. The Mann–Whitney U-test was used to test the difference between the groups defined by dichotomous variables (e.g., males vs. females; exposed to passive smoking vs. not exposed, etc.). The Kruskal–Wallis H-test was used for groups defined by the categorical variable with more than two categories (e.g., low, medium or high AMD, etc.). χ2 or Fisher’s exact test was used if the absolute frequencies in each category were statistically different. A correlation analysis (Pearson’s r) was performed to test the linear relationship between 25(OH)D levels in different biological matrices and was quantified using the ELISA or CMIA analytical method.
For sensitivity analyses, we firstly stratified the correlation analysis by sex and secondly compared 25(OH)D medians (Kruskal–Wallis test) across the status of different vitamin D levels (deficiency vs. sufficiency), which are defined according to recommendations provided by the Institute of Medicine [14] and the Endocrine Society [13]. The significance level was set at 5%.

3. Results

3.1. Sample Population Characteristics

As reported in Table 1, a sample of 62 young people (53.2% females) aged between 25 and 44 years (31.5 ± 5.1 years) was enrolled. Most participants were normal-weight adults (71%), around 21% were overweight, and none were obese. Around 52% of the study sample were workers, and 35.5% were students, but they shared similar working conditions as they had been recruited among health professionals and residents of a university department. In line with the inclusion criteria, none were active smokers, while around 15% of the participants reported exposure to passive smoking. According to the scoring obtained with the Medi-Lite questionnaire, only 22.6% of the enrolled subjects sample reported a high AMD. Concerning sun exposure-related behaviours, 73% of the participants routinely used sun cream, although most of them (54.8%) declared short habitual sun exposure periods lasting for less than 15 min per day. Regarding phototypes, participants with very light skin (phototype I) or light skin and brown hair (phototype II) made up the largest proportion of the sample, accounting for around 36% and 37% of the subjects, respectively.

3.2. Serum 25(OH)D Quantification by CMIA (Benchmark Measure)

Serum 25(OH)D levels, quantified by the CMIA, are reported in Table 2. As previously mentioned, this measurement was considered the benchmark in our study because the CMIA method is commonly and officially used to diagnose vitamin D deficiency. Frequencies of deficiency greatly differed according to IOM and ES cut-offs, with the latter classifying around 69% of the subjects as deficient. No differences were detected among CMIA-assessed serum levels of 25(OH)D between males and females (p = 0.70) or discrepancies among deficiency frequencies according to IOM or ES (p = 0.76 and 0.73, respectively).

3.3. Serum, Urine, and Saliva 25(OH)D Quantification by ELISA

Table 3 summarises the 25(OH)D levels assessed in serum, saliva, and urine by ELISA. Median 25(OH)D levels in urine were higher in females compared with those measured in males (p = 0.04). Between-group comparisons highlighted lower median 25(OH)D levels in saliva in participants reporting exposure to passive tobacco smoking (13.1 ng/mL) compared to those not passively exposed (19.2 ng/mL) (Mann–Whitney U-test p-value = 0.02). No other statistically significant differences were detected in 25(OH)D levels quantified in all biological matrices and both analytical methods.

3.4. Correlation Between the Benchmark Measure and 25(OH)D, Measured by ELISA in Serum, Urine, and Saliva

As reported in Table 4, urine 25(OH)D levels quantified by ELISA were significantly and positively correlated with the benchmark measure of vitamin D and serum 25(OH)D quantified by CMIA (Pearson’s r = 0.44, p = 0.05). No other correlations were observed among different matrices and analytical methods.
The same results were observed after sex stratification (Figure 1). However, the statistical significance was only confirmed in women whose urine 25(OH)D levels were more strongly correlated with the benchmark quantification (Pearson’s r = 0.62; p = 0.04).
Additionally, we further analysed if urine 25(OH)D levels varied by vitamin D deficiency status and observed that females with a Vitamin D deficiency, according to the ES, had lower median urine 25(OH)D (1.24 ng/mg creatinine) compared to those without deficiency (2.69 ng/mg creatinine) (Mann–Whitney U-test p-value = 0.04) (Figure 2). We did not observe the same results for men (p-value = 0.39) or in subjects classified according to the IOM cut-offs.

4. Discussion

The present study suggests a positive and statistically significant linear correlation between 25(OH)D levels, measured using a cost-effective method (ELISA) in non-invasive samples (urine), and a benchmark measure of vitamin D status (i.e., serum 25(OH)D quantified by CMIA). After stratifying by sex, this relationship was confirmed only in women and not in men. Additionally, we observed that median urine 25(OH)D levels were lower in women classified as vitamin D-deficient based on the ES threshold applied to serum benchmark quantifications. Serum and saliva 25(OH)D levels, quantified by ELISA, were not correlated with the benchmark measure or with each other.
Median serum 25(OH)D levels, quantified by CMIA, were comparable, although slightly higher, compared to those reported by Lahoz and colleagues [34], who measured 25(OH)D using the same analytical method in a group of individuals without vitamin D supplementation. The vast majority of previous studies employed a wide range of analytical techniques to assess serum 25(OH)D levels, mainly including ELISA [20,22,23,27,35,36,37,38,39], electrochemiluminescence enzyme immunoassays [18,39,40,41], chromatographic techniques [29,42,43,44], radioimmunoassay [45], and other techniques [19,30]. In these studies, the reported average/median serum 25(OH)D levels varied widely and were only partly comparable to our results, particularly those obtained from chromatographic or radioimmunoassay techniques, which ranged from 18.7 to 32.8 ng/mL and from 24.7 to 36.4 ng/mL, respectively. In our study, when applying the IOM and ES recommendations to categorise the distribution of serum 25(OH)D levels derived from CMIA measurements, 25.8% and 67.7% of participants were classified as vitamin D-deficient, respectively. The observed prevalence of deficiency was lower than the age-specific (19–45 years) and latitude-specific prevalence reported by Cui and colleagues in their systematic analyses, which included more than 150 studies [46]. Similarly, compared with the deficiency prevalence observed in the Italian adult population (i.e., 33% in the study by Lippi and colleagues [47]; 36% in the study by Lippi and colleagues [48]), we observed a lower deficiency prevalence. Another Italian study on a sample of 18,131 Italians from the general population reported a vitamin D deficiency prevalence of 35.47%, with a significantly higher prevalence in men than in women (40.12% vs. 33.10%) [49]. While the deficiency prevalence reported by Bani and colleagues was higher than the one observed in our study, their finding of a higher prevalence in men than in women is consistent with our results.
To date, the quantification of 25(OH)D in urine has been scarcely studied. In general, urinary vitamin D levels are quantified by chromatographic techniques [24,28,29], while some authors have reported measurements via immunoassays [24,28,29], and some authors have reported measurements using immunoassays [25,26] and the NLucVDR assay system [29,30]. Salivary 25(OH)D quantifications are more frequent than urinary quantifications and are typically performed using ELISA techniques, with reported results ranging widely from 36 pg/mL to 85.7 ng/mL. [21,23]. The mean salivary values observed in our study (i.e., 18.8 ng/mL) closely align with those reported by Sari et al. (i.e., 16.54 ng/mL) [22], obtained from a sample of 56 healthy Indonesian adults aged 18 to 60 years.
Our main finding was a statistically significant and positive correlation between serum benchmark 25(OH)D levels and urinary ones. This result is inconsistent with the findings from Kushioka and colleagues [29,30], who suggested that urine analysis may be useful for predicting serum 25(OH)D3 levels and developed a specific analytical method, the NLucVDR assay. Similarly, a recent research letter highlighted that urine analysis could also serve as a diagnostic tool for vitamin D detection, particularly in aiding clinicians in managing urinary tract infections [26]. In contrast, we did not observe correlations between vitamin D levels in serum and saliva. This finding aligns with some previous studies [19,23], but it is in contrast with others [18,20,21,38]. Notably, the detected correlation was based on a limited number of observations, as only around 34% of the urine samples (n = 21) had 25(OH)D levels above the limit of detection (LOD).
Regardless of the biological matrix, women, on average, exhibited higher 25(OH)D levels than men. This finding is consistent with previous research on sex-specific vitamin D levels involving adults from Saudi Arabia [50], Chile [51], and India [52] but contrasts with studies reporting a greater vulnerability to vitamin D deficiency in women [46,53,54,55]. Overall, sex appears to play a crucial role in the differences in circulating 25(OH)D levels between men and women, as well as in the degree of correlation between serum and urine 25(OH)D measurements. Reproductive hormones may affect the expression of genes related to vitamin D metabolisms [56] due to estrogenic therapies [57]. Previous studies have reported higher total 25OHD concentrations in women who take oral contraceptives containing oestrogen, possibly because of an oestrogen-related increased hepatic hydroxylation of vitamin D [58], although this phenomenon was not confirmed in girls not taking hormonal contraceptives [59]. In addition, VDBP concentration is generally higher in women than in men [60], and, together with the hormone asset, this element may partly explain the higher vitamin D levels observed in young women. Furthermore, evidence from in vivo studies suggests that when compared to male mice, female mice can synthesise vitamin D in their skin more efficiently [61]. On the whole, sex-dependent influences on vitamin D levels and interplay with other molecules and/or therapies seem to be critical [62] and should always be taken into account.
In this study, we did not observe any correlation between serum 25(OH)D concentration measured by ELISA and serum CMIA. Although seemingly surprising, this result suggests that a matrix effect could have strongly influenced the ability of the binding agent to efficiently associate with 25(OH)D [63], endorsing the need for the improvement of the analytical method by adding purification or precipitation steps. As one of the main limitations, we did not apply any pre-treatment step. This should be intended as an important step for future research on this topic, particularly regarding urine samples with 25(OH)D concentrations that might be very low, thus requiring additional method optimisation, both in terms of deconjugation and the breakdown of the carrier proteins’ bond. Given the explorative nature of the present study, we opted for a simpler first approach that, given the promising results, deserves future in-depth implementations. Another potential limitation is the sample size, which, however, was extremely homogeneous in terms of several potential confounders (e.g., age, sex, education level, ponderal status, etc.). Moving on to the strengths, our study is one of the few existing works on vitamin D quantification in emerging biological matrices such as urine and saliva. Two analytical methods (ELISA and CMIA) were used, and the correlation among their results was investigated in a sample of young adults.

5. Conclusions

Although far from definitive, our findings suggest the potential usefulness of non-invasively collected biological matrices, particularly urine, as potential novel media for quantifying 25(OH)D in young adults. The moderate correlation observed between 25(OH)D measured in urine (via ELISA) and serum (via CMIA) supports the feasibility of using non-invasive, cost-effective, and time-efficient analyses to advance vitamin D research. This approach is especially relevant for environmental epidemiology studies, which require higher subject compliance rates. Upon further validation, the quantification of 25(OH)D levels in urine could serve as a useful proxy for vitamin D status, particularly in non-diagnostic settings. It could be employed to explore the interaction between environmental and behavioural factors, such as air pollution and lifestyle habits, including smoking exposure and diet. Given that vitamin D deficiency is considered a contributing factor to various human diseases, supporting future epidemiological investigations into environmental and behavioural factors is essential. This would indirectly enhance our understanding of environmental exposures and chronic diseases. While this study provides preliminary insights, several aspects remain unexplored. Future research should focus on further improving the ELISA analytical methods, particularly through the evaluation of (1) conjugated and unconjugated 25(OH)D in urine; (2) free and total 25(OH)D levels in all biological matrices; and (3) potential cross-reactivity with other 25(OH)D metabolites.

Author Contributions

Conceptualisation, G.S. (Giulia Squillacioti); methodology, G.S. (Giulia Squillacioti), S.E.S., and S.L.; software, G.S. (Giulia Squillacioti) and V.L.; validation, G.M. and R.B.; formal analysis, G.S. (Giulia Squillacioti), S.E.S., and V.L.; investigation G.S. (Giulia Squillacioti) and S.E.S.; resources, G.S. (Giulia Squillacioti), G.M., and R.B.; data curation, V.L. and S.E.S.; writing—original draft preparation, G.S. (Giulia Squillacioti), S.E.S. and V.L.; writing—review and editing, G.S. (Giulia Squillacioti), S.E.S., V.L., F.G., M.P., G.S. (Giacomo Scaioli), M.M., S.L., G.M., and R.B.; visualisation, G.S. (Giulia Squillacioti); supervision, G.M. and R.B.; project administration, G.S. (Giulia Squillacioti) and R.B.; funding acquisition, G.S. (Giulia Squillacioti). All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the University of Turin and the “Grant for Internationalisation” programme. The APC was funded by R.B’s research funds.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Turin (Protocol n. 0654272, 24 October 2023).

Informed Consent Statement

Informed consent was obtained from all subjects involved in this study. Written informed consent was obtained from the patient(s) to publish this paper.

Data Availability Statement

The datasets presented in this article are not readily available because some restrictions to the dataset access have been declared in the documents approved by the University of Turin’s Bioethical Committee. Requests to access the datasets should be directed to giulia.squillacioti@unito.it.

Acknowledgments

We acknowledge all subjects for their participation.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Janoušek, J.; Pilařová, V.; Macáková, K.; Nomura, A.; Veiga-Matos, J.; da Silva, D.D.; Remião, F.; Saso, L.; Malá-Ládová, K.; Malý, J.; et al. Vitamin D: Sources, Physiological Role, Biokinetics, Deficiency, Therapeutic Use, Toxicity, and Overview of Analytical Methods for Detection of Vitamin D and Its Metabolites. Crit. Rev. Clin. Lab. Sci. 2022, 59, 517–554. [Google Scholar] [CrossRef] [PubMed]
  2. Aiello, G.; Lombardo, M.; Baldelli, S. Exploring Vitamin D Synthesis and Function in Cardiovascular Health: A Narrative Review. Appl. Sci. 2024, 14, 4339. [Google Scholar] [CrossRef]
  3. Jones, G.; Prosser, D.E.; Kaufmann, M. Cytochrome P450-Mediated Metabolism of Vitamin D. J. Lipid Res. 2014, 55, 13–31. [Google Scholar] [CrossRef]
  4. Jenkinson, C.; Desai, R.; McLeod, M.D.; Wolf Mueller, J.; Hewison, M.; Handelsman, D.J. Circulating Conjugated and Unconjugated Vitamin D Metabolite Measurements by Liquid Chromatography Mass Spectrometry. J. Clin. Endocrinol. Metab. 2022, 107, 435–449. [Google Scholar] [CrossRef]
  5. Schiffer, L.; Barnard, L.; Baranowski, E.S.; Gilligan, L.C.; Taylor, A.E.; Arlt, W.; Shackleton, C.H.L.; Storbeck, K.H. Human Steroid Biosynthesis, Metabolism and Excretion Are Differentially Reflected by Serum and Urine Steroid Metabolomes: A Comprehensive Review. J. Steroid Biochem. Mol. Biol. 2019, 194, 105439. [Google Scholar] [CrossRef] [PubMed]
  6. Tuckey, R.C.; Cheng, C.Y.S.; Slominski, A.T. The Serum Vitamin D Metabolome: What We Know and What Is Still to Discover. J. Steroid Biochem. Mol. Biol. 2019, 186, 4–21. [Google Scholar] [CrossRef]
  7. Watkins, R.R.; Yamshchikov, A.V.; Lemonovich, T.L.; Salata, R.A. The Role of Vitamin D Deficiency in Sepsis and Potential Therapeutic Implications. J. Infect. 2011, 63, 321–326. [Google Scholar] [CrossRef]
  8. Karras, S.N.; Wagner, C.L.; Castracane, V.D. Understanding Vitamin D Metabolism in Pregnancy: From Physiology to Pathophysiology and Clinical Outcomes. Metabolism 2018, 86, 112–123. [Google Scholar] [CrossRef]
  9. Zhao, S.; Qian, F.; Wan, Z.; Chen, X.; Pan, A.; Liu, G. Vitamin D and Major Chronic Diseases. Trends Endocrinol. Metab. 2024, 35, 1050–1061. [Google Scholar] [CrossRef]
  10. D’avolio, A.; Avataneo, V.; Manca, A.; Cusato, J.; De Nicolò, A.; Lucchini, R.; Keller, F.; Cantù, M. 25-Hydroxyvitamin D Concentrations Are Lower in Patients with Positive PCR for SARS-CoV-2. Nutrients 2020, 12, 1359. [Google Scholar] [CrossRef]
  11. Tsuprykov, O.; Chen, X.; Hocher, C.F.; Skoblo, R.; Yin, L.; Hocher, B. Why Should We Measure Free 25(OH) Vitamin D? J. Steroid Biochem. Mol. Biol. 2018, 180, 87–104. [Google Scholar] [CrossRef] [PubMed]
  12. Bikle, D.D.; Schwartz, J. Vitamin D Binding Protein, Total and Free Vitamin D Levels in Different Physiological and Pathophysiological Conditions. Front. Endocrinol. 2019, 10, 317. [Google Scholar] [CrossRef] [PubMed]
  13. Holick, M.F.; Binkley, N.C.; Bischoff-Ferrari, H.A.; Gordon, C.M.; Hanley, D.A.; Heaney, R.P.; Murad, M.H.; Weaver, C.M. Evaluation, Treatment, and Prevention of Vitamin D Deficiency: An Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab. 2011, 96, 1911–1930. [Google Scholar] [CrossRef]
  14. Owens, D.J.; Allison, R.; Close, G.L. Vitamin D and the Athlete: Current Perspectives and New Challenges. Sports Med. 2018, 48, 3–16. [Google Scholar] [CrossRef]
  15. Farrell, C.J.L.; Soldo, J.; McWhinney, B.; Bandodkar, S.; Herrmann, M. Impact of Assay Design on Test Performance: Lessons Learned from 25-Hydroxyvitamin D. Clin. Chem. Lab. Med. 2014, 52, 1579–1587. [Google Scholar] [CrossRef] [PubMed]
  16. Fraser, W.D.; Milan, A.M. Vitamin D Assays: Past and Present Debates, Difficulties, and Developments. Calcif. Tissue Int. 2013, 92, 118–127. [Google Scholar] [CrossRef]
  17. Herrmann, M.; Harwood, T.; Gaston-Parry, O.; Kouzios, D.; Wong, T.; Lih, A.; Jimenez, M.; Janu, M.; Seibel, M.J. A New Quantitative LC Tandem Mass Spectrometry Assay for Serum 25-Hydroxy Vitamin D. Steroids 2010, 75, 1106–1112. [Google Scholar] [CrossRef]
  18. Bahramian, A.; Falsafi, P.; Abbasi, T.; Ghanizadeh, M.; Abedini, M.; Kavoosi, F.; Kouhsoltani, M.; Noorbakhsh, F.; Dabbaghi Tabriz, F.; Rajaeih, S.; et al. Comparing Serum and Salivary Levels of Vitamin D in Patients with Recurrent Aphthous Stomatitis and Healthy Individuals. J. Dent. 2018, 19, 295. [Google Scholar]
  19. Fairney, A.; Saphier, P.W. Studies on the Measurement of 25-Hydroxy Vitamin D in Human Saliva. Br. J. Nutr. 1987, 57, 13–25. [Google Scholar] [CrossRef]
  20. Hussein, A.S.; Almoudi, M.M.; Abu-Hassan, M.I.; Schroth, R.J.; Saripudin, B.; Mohamad, M.S.F. Serum and Saliva 25(OH)D Levels in Relation to Dental Caries in Young Children. J. Clin. Pediatr. Dent. 2021, 45, 414–420. [Google Scholar] [CrossRef]
  21. Mirzaii-Dizgah, M.R.; Mirzaii-Dizgah, M.H.; Mirzaii-Dizgah, I.; Koshkzari, R. The Association between Saliva and Serum Vitamin D with Knee Osteoarthritis. J. Nutr. Food Secur. 2022, 8, 108–113. [Google Scholar] [CrossRef]
  22. Sari, D.K.; Sari, L.M.; Laksmi, L.I. Farhat The Moderate Correlation Between 25(OH)D Serum and Saliva in Healthy People with Low Vitamin D Intake. Int. J. Gen. Med. 2021, 14, 841–850. [Google Scholar] [CrossRef] [PubMed]
  23. Abdolsamadi, H.; Vahedi, M.; Fariba, F.; Soltanian, A.; Avval, M.Z.; Hosseini, A. Comparative Study of Salivary and Serum Levels of Vitamin D in Patients with a History of High Blood Pressure and Healthy People. J. Mol. Biol. Res. 2018, 8, p101. [Google Scholar] [CrossRef]
  24. Ogawa, S.; Ooki, S.; Shinoda, K.; Higashi, T. Analysis of Urinary Vitamin D3 Metabolites by Liquid Chromatography/Tandem Mass Spectrometry with ESI-Enhancing and Stable Isotope-Coded Derivatization. Anal. Bioanal. Chem. 2014, 406, 6647–6654. [Google Scholar] [CrossRef]
  25. Sato, K.A.; Gray, R.W.; Lemann, J. Urinary Excretion of 25-Hydroxyvitamin D in Health and the Nephrotic Syndrome. J. Lab. Clin. Med. 1982, 99, 325–330. [Google Scholar]
  26. Piccolini, A.; Grizzi, F.; Monari, M.; Hegazi, M.A.A.A.; Buffi, N.M.; Casale, P.; Fasulo, V.; Moretto, S.; Cella, L.; Vota, P.; et al. Preliminary Findings on Vitamin D 25-OH Levels in Urine Analysis: Implications for Clinical Practice. BJU Int. 2024, 134, 561–563. [Google Scholar] [CrossRef]
  27. McWhorter, C.A.; Mead, M.J.; Rodgers, M.D.; Ebeling, M.D.; Shary, J.R.; Gregoski, M.J.; Newton, D.A.; Baatz, J.E.; Hollis, B.W.; Hewison, M.; et al. Predicting Comorbidities of Pregnancy: A Comparison between Total and Free 25(OH)D and Their Associations with Parathyroid Hormone. J. Steroid Biochem. Mol. Biol. 2023, 235, 106420. [Google Scholar] [CrossRef] [PubMed]
  28. Tamblyn, J.A.; Jenkinson, C.; Larner, D.P.; Hewison, M.; Kilby, M.D. Serum and Urine Vitamin D Metabolite Analysis in Early Preeclampsia. Endocr. Connect. 2018, 7, 199–210. [Google Scholar] [CrossRef]
  29. Kushioka, T.; Mano, H.; Matsuoka, S.; Nishikawa, M.; Yasuda, K.; Ikushiro, S.; Sakaki, T. Analysis of Vitamin D Metabolites in Biological Samples Using a Nanoluc-Based Vitamin D Receptor Ligand Sensing System: NLucVDR. J. Steroid Biochem. Mol. Biol. 2023, 233, 106367. [Google Scholar] [CrossRef]
  30. Kushioka, T.; Mano, H.; Matsuoka, S.; Nishikawa, M.; Yasuda, K.; Ikushiro, S.; Sakaki, T. Association between Serum 25-Hydroxyvitamin D Concentrations and Urinary Vitamin D Metabolite Concentrations Measured by the NLucVDR Assay. J. Steroid Biochem. Mol. Biol. 2025, 247, 106678. [Google Scholar] [CrossRef]
  31. Simundic, A.M.; Bölenius, K.; Cadamuro, J.; Church, S.; Cornes, M.P.; Van Dongen-Lases, E.C.; Eker, P.; Erdeljanovic, T.; Grankvist, K.; Guimaraes, J.T.; et al. Joint EFLM-COLABIOCLI Recommendation for Venous Blood Sampling. Clin. Chem. Lab. Med. 2018, 56, 2015–2038. [Google Scholar] [CrossRef] [PubMed]
  32. Giustina, A.; Bilezikian, J.P.; Adler, R.A.; Banfi, G.; Bikle, D.D.; Binkley, N.C.; Bollerslev, J.; Bouillon, R.; Brandi, M.L.; Casanueva, F.F.; et al. Consensus Statement on Vitamin D Status Assessment and Supplementation: Whys, Whens, and Hows. Endocr. Rev. 2024, 45, 625–654. [Google Scholar] [CrossRef] [PubMed]
  33. Sofi, F.; Dinu, M.; Pagliai, G.; Marcucci, R.; Casini, A. Validation of a Literature-Based Adherence Score to Mediterranean Diet: The MEDI-LITE Score. Int. J. Food Sci. Nutr. 2017, 68, 757–762. [Google Scholar] [CrossRef]
  34. Lahoz, R.; Sánchez, J.P.; Górriz, S.; Calmarza, P. Comparative Study of Two Immunoassays Used for the Determination of Serum Vitamin D. Pract. Lab. Med. 2021, 26, e00242. [Google Scholar] [CrossRef]
  35. Ahmed, M.A.; Al-Nafeesah, A.; AlEed, A.; Adam, I. Serum Level of 25-Hydroxyvitamin D and Symptoms of Pica Among Adolescent School Children in Northern Sudan: A Cross-Sectional Study. Glob. Pediatr. Health 2024, 11, 2333794X241242564. [Google Scholar] [CrossRef]
  36. Bakhuraysah, M.M.; Gharib, A.F.; Hassan, A.F.; Al Harthi, G.K.; Al Thobaiti, R.F.; Al Adwani, M.M.; Alharbi, A.D.; Alzahrani, A.S.; Alsubei, K.M.; Al-Asiri, R.F. Novel Insight Into the Relationship of Vitamin D Hydroxylase and Vitamin D With Obesity in Patients With Type 2 Diabetes Mellitus. Cureus 2023, 15. [Google Scholar] [CrossRef] [PubMed]
  37. He, C.-S.; Gleeson, M.; Fraser, W.D. Measurement of Circulating 25-Hydroxy Vitamin d Using Three Commercial Enzyme-Linked Immunosorbent Assay Kits with Comparison to Liquid Chromatography: Tandem Mass Spectrometry Method. ISRN Nutr. 2013, 2013, 723139. [Google Scholar] [CrossRef]
  38. Sari, D.K.; Sari, L.M.; Laksmi, L.I.; Farhat, F. The Use of 25-Hydroxyvitamin D Saliva Test to Replace Vitamin D Serum Blood Test in Healthy People. Open Access Maced. J. Med. Sci. 2021, 9, 40–43. [Google Scholar] [CrossRef]
  39. Mantecón, L.; Agustina Alonso, M.; Moya, V.; Andrés, A.G.; Avello, N.; Martínez-Morillo, E.; Santos, F. Marker of Vitamin D Status in Healthy Children: Free or Total 25-Hydroxyvitamin D? PLoS ONE 2018, 13, e0202237. [Google Scholar] [CrossRef]
  40. Arshad, S.; Zaidi, S.J.A. Vitamin D Levels among Children, Adolescents, Adults, and Elders in Pakistani Population: A Cross-Sectional Study. BMC Public. Health 2022, 22, 2040. [Google Scholar] [CrossRef]
  41. Banerjee, S.; Basu, S.; Akhtar, S.; Sinha, R.; Sen, A.; Sengupta, J. Free Vitamin D Levels in Steroid-Sensitive Nephrotic Syndrome and Healthy Controls. Pediatr. Nephrol. 2020, 35, 447–454. [Google Scholar] [CrossRef] [PubMed]
  42. Binkley, N.; Novotny, R.; Krueger, D.; Kawahara, T.; Daida, Y.G.; Lensmeyer, G.; Hollis, B.W.; Drezner, M.K. Low Vitamin D Status despite Abundant Sun Exposure. J. Clin. Endocrinol. Metab. 2007, 92, 2130–2135. [Google Scholar] [CrossRef]
  43. Bowles, S.D.; Basu, S.; Ranchordas, M.K.; Simper, T.; Lynn, A. Circulating Total 25(OH)D and Calculated Free 25(OH)D in Professional Academy Footballers at a Northerly Latitude in the UK. Biol. Sport. 2024, 41, 201–208. [Google Scholar] [CrossRef] [PubMed]
  44. Sollid, S.T.; Hutchinson, M.Y.S.; Berg, V.; Fuskevåg, O.M.; Figenschau, Y.; Thorsby, P.M.; Jorde, R. Effects of Vitamin D Binding Protein Phenotypes and Vitamin D Supplementation on Serum Total 25(OH)D and Directly Measured Free 25(OH)D. Eur. J. Endocrinol. 2016, 174, 445–452. [Google Scholar] [CrossRef]
  45. Bolland, M.J.; Grey, A.B.; Ames, R.W.; Horne, A.M.; Mason, B.H.; Wattie, D.J.; Gamble, G.D.; Bouillon, R.; Reid, I.R. Age-, Gender-, and Weight-Related Effects on Levels of 25-Hydroxyvitamin D Are Not Mediated by Vitamin D Binding Protein. Clin. Endocrinol. 2007, 67, 259–264. [Google Scholar] [CrossRef] [PubMed]
  46. Cui, A.; Zhang, T.; Xiao, P.; Fan, Z.; Wang, H.; Zhuang, Y. Global and Regional Prevalence of Vitamin D Deficiency in Population-Based Studies from 2000 to 2022: A Pooled Analysis of 7.9 Million Participants. Front. Nutr. 2023, 10, 1070808. [Google Scholar] [CrossRef]
  47. Lippi, G.; Montagnana, M.; Meschi, T.; Borghi, L. Vitamin D Concentration and Deficiency across Different Ages and Genders. Aging Clin. Exp. Res. 2012, 24, 548–551. [Google Scholar] [CrossRef]
  48. Lippi, G.; Nouvenne, A.; Ticinesi, A.; Bonelli, P.; Salvagno, G.L.; Cervellin, G.; Guidi, G.C. The Burden of Vitamin D Deficiency in a Mediterranean Country without a Policy of Food Fortification. Acta Biomed. 2015, 86, 59–62. [Google Scholar]
  49. Bani, P.; Cingari, S.; Mathis, B. Study on the Prevalence of Vitamin D Deficiency in the Population of Canton Ticino. Riv. Ital. Della Med. Lab. 2022, 18, 18–21. [Google Scholar] [CrossRef]
  50. AlQuaiz, A.J.M.; Kazi, A.; Fouda, M.; Alyousefi, N. Age and Gender Differences in the Prevalence and Correlates of Vitamin D Deficiency. Arch. Osteoporos. 2018, 13, 49. [Google Scholar] [CrossRef]
  51. Vallejo, M.S.; Blümel, J.E.; Arteaga, E.; Aedo, S.; Tapia, V.; Araos, A.; Sciaraffia, C.; Castelo-Branco, C. Gender Differences in the Prevalence of Vitamin D Deficiency in a Southern Latin American Country: A Pilot Study. Climacteric 2020, 23, 410–416. [Google Scholar] [CrossRef] [PubMed]
  52. Sanghera, D.K.; Sapkota, B.R.; Aston, C.E.; Blackett, P.R. Vitamin D Status, Gender Differences, and Cardiometabolic Health Disparities. Ann. Nutr. Metab. 2017, 70, 79–87. [Google Scholar] [CrossRef] [PubMed]
  53. Verdoia, M.; Schaffer, A.; Barbieri, L.; Di Giovine, G.; Marino, P.; Suryapranata, H.; De Luca, G. Impact of Gender Difference on Vitamin D Status and Its Relationship with the Extent of Coronary Artery Disease. Nutr. Metab. Cardiovasc. Dis. 2015, 25, 464–470. [Google Scholar] [CrossRef]
  54. Al-Hetar, M.; Al-Goshae, H.; Wahab, N.; Abdulghani, M.; Faez Baobaid, M.; Al-matary, A.M.; Al-hetar, Q.; Alezzy, M.; Senan, D.; Ruhi, S. Prevalence, Predictors, and Gender-Based Risk Factors of Vitamin D Deficiency: A Retrospective Cross-Sectional Study. J. Angiother. 2024, 8, 1–9. [Google Scholar] [CrossRef]
  55. Wierzbicka, A.; Oczkowicz, M. Sex Differences in Vitamin D Metabolism, Serum Levels and Action. Br. J. Nutr. 2022, 128, 2115–2130. [Google Scholar] [CrossRef] [PubMed]
  56. Zhao, D.; Ouyang, P.; de Boer, I.H.; Lutsey, P.L.; Farag, Y.M.K.; Guallar, E.; Siscovick, D.S.; Post, W.S.; Kalyani, R.R.; Billups, K.L.; et al. Serum Vitamin D and Sex Hormones Levels in Men and Women: The Multi-Ethnic Study of Atherosclerosis (MESA). Maturitas 2017, 96, 95–102. [Google Scholar] [CrossRef]
  57. Harmon, Q.E.; Umbach, D.M.; Baird, D.D. Use of Estrogen-Containing Contraception Is Associated With Increased Concentrations of 25-Hydroxy Vitamin D. J. Clin. Endocrinol. Metab. 2016, 101, 3370–3377. [Google Scholar] [CrossRef]
  58. Harris, S.S.; Dawson-Hughes, B. The Association of Oral Contraceptive Use with Plasma 25-Hydroxyvitamin D Levels. J. Am. Coll. Nutr. 1998, 17, 282–284. [Google Scholar] [CrossRef]
  59. Knight, J.A.; Wong, J.; Blackmore, K.M.; Raboud, J.M.; Vieth, R. Vitamin D Association with Estradiol and Progesterone in Young Women. Cancer Causes Control 2010, 21, 479–483. [Google Scholar] [CrossRef]
  60. Lagunova, Z.; Porojnicu, A.C.; Lindberg, F.; Hexeberg, S.; Moan, J. The Dependency of Vitamin D Status on Body Mass Index, Gender, Age and Season. Anticancer. Res. 2009, 29, 3713–3720. [Google Scholar] [CrossRef]
  61. Rebel, H.; Dingemanse-Van Der Spek, C.; Salvatori, D.; Van Leeuwen, J.P.T.M.; Robanus-Maandag, E.C.; De Gruijl, F.R. UV Exposure Inhibits Intestinal Tumor Growth and Progression to Malignancy in Intestine-Specific Apc Mutant Mice Kept on Low Vitamin D Diet. Int. J. Cancer 2015, 136, 271–277. [Google Scholar] [CrossRef] [PubMed]
  62. Crescioli, C.; Minisola, S. Vitamin D: Autoimmunity and Gender. Curr. Med. Chem. 2017, 24, 2671–2686. [Google Scholar] [CrossRef] [PubMed]
  63. Hollis, B.W. Assessment and Interpretation of Circulating 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D in the Clinical Environment. Endocrinol. Metab. Clin. N. Am. 2010, 39, 271–286. [Google Scholar] [CrossRef] [PubMed]
Biomolecules 15 00560 g001
Figure 1. Correlation between urine and serum 25(OH)D (n = 21) measured by Enzyme-Linked Immunosorbent Assay (ELISA) and chemiluminescent immunoassay with microparticle capture (CMIA) methods, stratified by sex.
Figure 1. Correlation between urine and serum 25(OH)D (n = 21) measured by Enzyme-Linked Immunosorbent Assay (ELISA) and chemiluminescent immunoassay with microparticle capture (CMIA) methods, stratified by sex.
Biomolecules 15 00560 g001
Biomolecules 15 00560 g002
Figure 2. Urine 25(OH)D levels measured by Enzyme-Linked Immunosorbent Assay (ELISA) according to vitamin D deficiency status in females. The asterisk represents values that are >3.0 IQR above the third quartile, while the circle represents values that are <1.5 IQR below the first quartile.
Figure 2. Urine 25(OH)D levels measured by Enzyme-Linked Immunosorbent Assay (ELISA) according to vitamin D deficiency status in females. The asterisk represents values that are >3.0 IQR above the third quartile, while the circle represents values that are <1.5 IQR below the first quartile.
Biomolecules 15 00560 g002
Table 1. Sample population characteristics.
Table 1. Sample population characteristics.
CharacteristicsOverall
62 (100%)		Females
33 (53.2%)		Males
29 (46.8%)
Age, mean (SD) [years] 31.5 (5.1)30.4 (4.1)32.8 (5.9)
BMI categories, n%
Normal weight44 (71.0)30 (48.4)14 (22.6)
Overweight13 (20.9)2 (3.2)11 (17.7)
Missing data5 (8.1)1 (1.6)4 (6.5)
Occupation, n (%)
Students22 (35.5)12 (19.4)10 (16.1)
Workers32 (51.6)18 (29.0)14 (22.6)
Missing data8 (12.9)3 (4.8)5 (8.1)
AMD, n (%)
Low (1st tertile)34 (54.8)20 (32.3)14 (22.6)
Medium (2nd tertile)9 (14.5)6 (9.7)3 (4.8)
High (3rd tertile)14 (22.6)6 (9.7)8 (12.9)
Missing data5 (8.1)1 (1.6)4 (6.5)
Tobacco smoking exposure, n (%)
Passive smokers9 (14.5)4 (6.5)5 (8.1)
Missing data5 (8.1)1 (1.6)4 (6.5)
Sun exposure behaviour, n (%)
Sun cream users45 (72.6)26 (41.9)19 (30.7)
Missing data5 (8.1)6 (9.7)4 (6.5)
Sun exposure < 15 min/day34 (54.8)22 (35.5)12 (19.4)
Missing data8 (12.9)2 (3.2)6 (9.7)
Phototype, n (%)
I. very light skin22 (35.5)15 (24.2)7 (11.3)
II. light eyes and skin23 (37.1)11 (17.7)12 (19.4)
III. light skin and brown hair5 (8.1)2 (3.2)3 (4.8)
IV. olive skin7 (11.3)4 (6.5)3 (4.8)
Missing data5 (8.1)1 (1.6)4 (6.5)
SD = standard deviation; BMI = Body Mass Index; AMD = adherence to the Mediterranean Diet.
Table 2. Serum 25(OH)D quantification by CMIA (benchmark measure).
Table 2. Serum 25(OH)D quantification by CMIA (benchmark measure).
Vitamin D Benchmark Levels (CMIA)Overall
62 (100%)		Females
33 (53.2%)		Males
29 (46.8%)		p-Value
Serum 25(OH)D, mean (SD)
ng/mL26.1 (9.1)26.6 (9.6)25.6 (8.7)0.70 a
nmoL/L65.3 (22.7)66.4 (23.9)64.1 (21.7)0.70 a
Serum 25(OH)D, median (IQR)
ng/mL25.2 (19.9–31.2)24.5 (20.8–32)25.2 (19.9–35.7)0.84 b
nmoL/L62.9 (49.8–78.0)61.3 (52–80)63 (49.8–77.5)0.84 b
Vitamin D deficiency, n (%)
IOM16 (25.8)8 (12.9)8 (12.9)0.76 c
ES42 (67.7)23 (37.1)19 (30.6)0.73 c
CMIA = chemiluminescent immunoassay with microparticle capture; SD = standard deviation; IOM = Institute of Medicine recommendations; ES = Endocrine Society recommendations. a t-test or b Mann–Whitney and c χ2 were used to test differences between females and males.
Table 3. ELISA quantifications of 25(OH)D in serum, saliva, and urine.
Table 3. ELISA quantifications of 25(OH)D in serum, saliva, and urine.
Vitamin D ELISA LevelsOverall
62 (100%)		Females
33 (53.2%)		Males
29 (46.8%)		p-Value
Serum 25(OH)D, mean (SD)
ng/mL6.54 (1.59)6.89 (3.31)6.13 (1.80)0.06
nmoL/L16.34 (3.99)17.24 (3.31)15.32 (4.50)0.06
Saliva 25(OH)D, mean (SD)
ng/mL18.79 (6.21)18.89 (6.44)18.68 (6.10)0.89
nmoL/L46.98 (15.53)47.23 (16.10)46.69 (15.14)0.89
Urine 25(OH)D, median (IQR)
ng/mg creatinine1.05 (2.57–0.59)1.38 (2.69–1.05)0.69 (0.96–0.47)0.04
nmoL/mmoL creatinine0.28 (0.68–0.18)0.44 (0.78–0.28)0.20 (0.26–0.14)0.04
ELISA = Enzyme-Linked Immunosorbent Assay; SD = standard deviation; IQR = interquartile range. t-test and Mann–Whitney were used to test differences between females and males.
Table 4. Correlation between benchmark 25(OH)D levels and those measured by ELISA in serum, saliva, and urine.
Table 4. Correlation between benchmark 25(OH)D levels and those measured by ELISA in serum, saliva, and urine.
25(OH)DSerum Benchmark (CMIA) [ng/mL]Serum ELISA [ng/mL]Urine ELISA [ng/mg Creatine]
Serum ELISA [ng/mL]r = −0.01
p = 0.94
n = 62
Urine ELISA [ng/mg crea]r = 0.44r = 0.11
p = 0.05p = 0.63
n = 21n = 21
Saliva ELISA [ng/mL]r = 0.10r = −0.12r = 0.05
p = 0.43p = 0.36p = 0.83
n = 62n =62n = 21
ELISA = Enzyme-Linked Immunosorbent Assay; CMIA = chemiluminescent immunoassay with microparticle capture. All p-values are derived from Pearson’s correlation analysis.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Squillacioti, G.; El Sherbiny, S.; Lettico, V.; Ghelli, F.; Panizzolo, M.; Scaioli, G.; Martella, M.; Limoncelli, S.; Mengozzi, G.; Bono, R. The Quantification of Vitamin D in Humans: A Promising, Non-Invasive and Cost-Effective Method to Measure 25-Hydroxyvitamin D. Biomolecules 2025, 15, 560. https://doi.org/10.3390/biom15040560

AMA Style

Squillacioti G, El Sherbiny S, Lettico V, Ghelli F, Panizzolo M, Scaioli G, Martella M, Limoncelli S, Mengozzi G, Bono R. The Quantification of Vitamin D in Humans: A Promising, Non-Invasive and Cost-Effective Method to Measure 25-Hydroxyvitamin D. Biomolecules. 2025; 15(4):560. https://doi.org/10.3390/biom15040560

Chicago/Turabian Style

Squillacioti, Giulia, Samar El Sherbiny, Veronica Lettico, Federica Ghelli, Marco Panizzolo, Giacomo Scaioli, Manuela Martella, Selene Limoncelli, Giulio Mengozzi, and Roberto Bono. 2025. "The Quantification of Vitamin D in Humans: A Promising, Non-Invasive and Cost-Effective Method to Measure 25-Hydroxyvitamin D" Biomolecules 15, no. 4: 560. https://doi.org/10.3390/biom15040560

APA Style

Squillacioti, G., El Sherbiny, S., Lettico, V., Ghelli, F., Panizzolo, M., Scaioli, G., Martella, M., Limoncelli, S., Mengozzi, G., & Bono, R. (2025). The Quantification of Vitamin D in Humans: A Promising, Non-Invasive and Cost-Effective Method to Measure 25-Hydroxyvitamin D. Biomolecules, 15(4), 560. https://doi.org/10.3390/biom15040560

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop