**Calcium, Vitamin D and Health**

Special Issue Editor **Luis Gracia-Marco**

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*Special Issue Editor* Luis Gracia-Marco University of Granada Spain

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This is a reprint of articles from the Special Issue published online in the open access journal *Nutrients* (ISSN 2072-6643) (available at: https://www.mdpi.com/journal/nutrients/special issues/calcium vitaminD health).

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## **Contents**



## **About the Special Issue Editor**

**Luis Gracia-Marco** is Senior Research Fellow in the Faculty of Sport Sciences at the University of Granada (Spain) and former Senior Lecturer at the University of Exeter (U.K.). He was awarded his Ph.D. in 2011 and has published more than 80 peer-reviewed Journal Citation Report (JCR) articles in the fields of body composition, sport sciences, and endocrinology and metabolism. He has supervised a number of B.S., M.S., and Ph.D. students in these areas. He has been involved in studies funded by the European Union and other international and competitive calls as principal investigator and researcher. Dr. Gracia-Marco also leads the bone and exercise research line in the PROFITH (PROmoting FITness and Health through physical activity) Research Group.

## *Editorial* **Calcium, Vitamin D, and Health**

#### **Luis Gracia-Marco**

PROFITH "PROmoting FITness and Health through Physical Activity" Research Group, Sport and Health University Research Institute (iMUDS), Department of Physical Education and Sports, Faculty of Sport Sciences, University of Granada, 18071 Granada, Spain; lgracia@ugr.es

Received: 27 January 2020; Accepted: 4 February 2020; Published: 6 February 2020

Calcium is the main mineral in the body. It is involved in a variety of structural and functional roles, but the maintenance of calcium homeostasis is perhaps the most studied function of vitamin D. This Special Issue of *Nutrients*, "Calcium, Vitamin D, and Health" contains 12 original publications and two reviews investigating the contribution of (mainly) vitamin D and calcium on relevant health outcomes in a variety of populations, which reflect the evolving and broad interests of research on this topic.

Three studies were published examining the association between vitamin D and body composition. Rabufetti et al. [1] observed that an increase in body fat percentage was a risk factor for 25-hydroxyvitamin D [25(OH)D] insufficiency in a healthy population of 1045 late adolescent males living in southern Switzerland. Abboud et al. [2], in their study on men and women with overweight/obese and undergoing a weight loss program, found greater weight loss, as well as a larger reduction in body mass index (BMI), and waist circumference in those with higher baseline 25(OH)D levels. Moreover, a similar effect was also observed in those with insufficient baseline 25(OH)D levels but supplemented with vitamin D3 for three months. In a study with middle-aged sedentary adults, De-la-O et al. [3] found negative associations between 1,25-dihydroxyvitamin D (1,25(OH)2D, also known as calcitriol, and BMI, lean mass index, and bone mineral density (BMD). The latter finding backs up the notion that 1,25(OH)2D increases bone resorption via stimulating intestinal calcium absorption after calcium intake.

Two other studies investigated the links between 25(OH)D and bone outcomes in young populations. Gil-Cosano et al. [4] revealed a mediating effect of muscular fitness on the relationship between 25(OH)D levels and BMD in children who were overweight/obese, whileRapun-Lopez et al. [5] showed similar bone remodeling in adolescent male cyclists than age-matched active controls over one year, but lower 25(OH)D. In adult and older women from the Chilean National Health Survey 2016–2017 (total *N* = 1931), Solis-Urra et al. [6] found a joint association of high sedentary time/passive commuting to be associated with 25(OH)D deficiency, even after controlling for sun exposure. This finding connects with the studies mentioned above [1–3] due to the proposed link between sedentary time and increased adiposity, as well as between adiposity and reduced 25(OH)D levels.

Libuda et al. [7] studied six single nucleotide polymorphisms (SNPs), which were genome-wide significantly associated with 25(OH)D concentrations in more than 79,000 subjects from the SUNLIGHT genome-wide association study (GWAS). However, they did not identify the potential role (from a genetics perspective) of 25(OH)D in the onset of depressive symptoms or broad depression. Multiple sclerosis (MS) has been negatively associated with BMD through various factors, and previous research has suggested that vitamin D could play a role in the pathogenesis of MS by possibly modulating T-lymphocyte subset differentiation. In this regard, Vlot et al. [8] studied the vitamin D-fibroblast-growth-factor-23 (FGF23) and measured multiple vitamin D metabolites and bone turnover markers in a cohort of MS patients and healthy controls. They found lower serum concentrations of total 25(OH)D, free 25(OH)D, free 1.25(OH)2D, and 24,25 dihydroxyvitamin D [24,25(OH)2D] in female MS patients compared with their healthy peers, while serum concentrations of vitamin D binding protein (VDBP) were higher in male MS patients, compared with male controls. This study strengthens the idea that a single measurement of total 25(OH)D may not be enough to

fully reflect all changes in vitamin D metabolism in MS patients. In a randomized clinical trial conducted in hypertensive adults, Francic et al. [9] did not support the routine measurement of 24,25 dihydroxycholecalciferol (24,25(OH)2D3) in order to individually optimize the dosage of vitamin D supplementation. Interestingly, the activity of 24-hydroxylase increased after vitamin D supplementation. In patients with postherpetic neuralgia (PHN), Chen et al. [10] showed a higher prevalence of hypovitaminosis D (as reflected by 25(OH)D levels) than in the controls, and those with hypovitaminosis D also had a lower vitamin D supplementation rate and greater pain intensity.

In healthy post-menopausal women, Reyes-Garcia et al. [11] investigated the response of serum 25(OH)D and its predictive factors after a 24-month dietary intervention with milk fortified with vitamin D and calcium. It was found that the improvement in 25(OH)D after the intervention was mainly dependent on the baseline levels of serum 25(OH)D and the percentage of body fat. The study by Jurimae et al. [12] is one of the few studies investigating the association between calcium and adiposity in young populations, and found inverse associations between dietary calcium intake and total body and abdominal adiposity in healthy male adolescents.

Finally, two timely reviews were included in this Special Issue. Brandao-Lima et al. [13] conducted a systematic review of randomized controlled trials aiming to discuss food fortification as a strategy for maintenance or recovery of nutritional status related to vitamin D in children. Marino and Misra [14], in their review, discussed the biological effects of vitamin D beyond the skeleton, using evidence from randomized controlled trials and meta-analyses.

The present Special Issue provides a short summary of the progress on the topic of calcium, vitamin D, and human health in different populations, which will be of interest from a clinical and public health perspective. It also underlines the current limitations and the necessity of more powerful study designs to further advance in the knowledge.

**Funding:** This research received no external funding.

**Acknowledgments:** L.G.-M. is supported by "La Caixa" Foundation within the Junior Leader fellowship programme (ID 100010434).

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Vitamin D Status Among Male Late Adolescents Living in Southern Switzerland: Role of Body Composition and Lifestyle**

**Andrea Rabufetti 1,**†**, Gregorio P. Milani 1,2,3,\*,**†**, Sebastiano A. G. Lava 4, Valeria Edefonti 3, Mario G. Bianchetti 5, Andreas Stettbacher 6, Franco Muggli <sup>6</sup> and Giacomo Simonetti 1,5**


Received: 27 September 2019; Accepted: 5 November 2019; Published: 11 November 2019

**Abstract:** Background: Poor vitamin D status is a worldwide health problem. Yet, knowledge about vitamin D status among adolescents in Southern Europe is limited. This study investigated concentrations and modulating factors of vitamin D in a healthy population of male late adolescents living in Southern Switzerland. Methods: All apparently healthy subjects attending for the medical evaluation before the compulsory military service in Southern Switzerland during 2014-2016 were eligible. Dark-skin subjects, subjects on vitamin D supplementation or managed with diseases or drugs involved in vitamin D metabolism were excluded. Anthropometric measurements (body height, weight, fat percentage, mid-upper arm and waist circumference) and blood sampling for total 25-hydroxy-vitamin D, total cholesterol and ferritin concentrations testing, were collected. Participants filled in a structured questionnaire addressing their lifestyle. Characteristics of the subjects with adequate (≥50 nmol/L–≤250 nmol/L) and insufficient (<50 nmol/L) vitamin D values were compared by Kruskal-Wallis test or χ<sup>2</sup> test. Odds ratios for 25-hydroxy-vitamin D insufficiency were calculated by univariate and AIC-selected multiple logistic regression models. Results: A total of 1045 subjects volunteered to participate in the study. Insufficient concentrations of vitamin D were detected in 184 (17%). The season of measurement was the most significant factor associated with vitamin D levels and approximately 40% of subjects presented insufficient vitamin D concentrations in winter. After model selection, body fat percentage, frequency and site of recreational physical activity, and the seasonality were significantly associated with the risk of vitamin D insufficiency. Conclusions: Among healthy male late adolescents in Southern Switzerland, about one every fourth subject presents a poor vitamin D status in non-summer seasons. Body fat percentage, frequent and outdoor recreational physical activity are modulating factors of vitamin D status in this population.

**Keywords:** macronutrients; sunlight; physical activity; season; body composition

#### **1. Introduction**

There are two natural sources of vitamin D: food and especially ultraviolet B radiation on the skin [1]. A limited number of foods naturally contain vitamin D. Fish (mostly fatty fish), egg yolk and liver are good sources of vitamin D3. On the other hand, vitamin D2 is contained in various wild mushrooms [1,2]. Among European adolescents, the natural vitamin D intake is low except for countries such as Poland and Norway, which is attributed to high consumption of fish [3].

The amount of cutaneous vitamin D3 synthesis depends on a number of factors, including time spent outdoors, latitude, season, ethnicity and use of sunscreen [1]. Vitamin D synthesis occurs for about half the year in northern regions above approximately 35◦ latitude [3,4]. Unsurprisingly, therefore, lower-than desired concentrations of total 25-hydroxy vitamin D have often been detected, especially during the fall and winter months, in countries such Canada, Ireland, the United Kingdom and the northern United States [3,4]. It would be assumed that, in the sunniest areas of the world, this problem would be uncommon. However, in Australia, Brazil, India, Iran, Lebanon and Saudi Arabia many adolescents were found to have lower-than-desired concentrations of vitamin D [3,4].

Limited information is available on vitamin D status in adolescents living in Southern Europe. The objective of the present analysis was to obtain reliable and comparable data on vitamin D status from a large population of late adolescents living in Southern Switzerland, the sunniest region of this country (latitude 46◦). The secondary aim was to investigate the role of a broad number of possibly relevant anthropometric, lifestyle and biochemical characteristics on vitamin D status in this population.

#### **2. Methods**

This investigation is part of the "CENERI study", a cross sectional study in healthy male adolescents living in Southern Switzerland to investigate risk factors for chronic diseases later in life. In Switzerland, ostensibly male citizens between 18 and 19 years of age have to undertake a medical evaluation before the compulsory military service in the Army [5]. All apparently healthy subjects attending for the medical evaluation before the compulsory military service in Southern Switzerland from January 2014 to December 2016 were eligible for the "CENERI study". Dark-skin subjects (Fitzpatrick skin phototype V or VI), subjects on supplementation with any form of vitamin D and subjects on treatment with anticonvulsant, glucocorticoid, antifungal, and anti-retroviral drugs or with any chronic endocrinologic or metabolic disease potentially affecting vitamin D metabolism, were excluded for the present analysis. Among the 4663 subjects who underwent the medical examination before the compulsory military service, 1045 (22%) Caucasians volunteered to participate in the study.

All measurements and data were collected in the same morning for each subject after an overnight fast. Beyond the routinely collected data on anthropometric measurements (body height and weight), participants were asked to answer a self-administered structured questionnaire addressing their main activity and lifestyle (especially recreational physical activity, smoking behavior and alcohol consumption). Body fat percentage, mid-upper arm and waist circumference were also measured. In addition, blood for total 25-hydroxy-vitamin D, total cholesterol and ferritin concentrations testing, was also collected.

Questions on lifestyle were structured as follows: (i) Frequency of recreational physical activity (never, 1 per week, 2–4 per week, 5–6 per week, every day), (ii) Duration of recreational physical activity session (≤1 h, >1–≤2 h, >2–≤3 h, >3 h), (iii) Site of recreational physical activity (indoor only, outdoor only, both indoor and outdoor), (iv) Frequency of alcohol consumption (never, 1 per week, 2 per week, 3–4 per week, 5–6 per week, every day), (v) Smoking (never, 1–10 cigarettes per day, 11–20 cigarettes per day, >20 cigarettes per day).

Subjects were weighed (wearing light clothes only) on a calibrated platform scale, with weight being rounded off to the nearest 0.1 kg. Standing height was measured barefooted to the nearest 0.1 cm. These measurements were used to calculate the body mass index. Mid-upper arm circumference was measured to the nearest 0.1 cm midway the acromion and the olecranon in the non-dominant arm. Waist circumference was measured to the nearest 0.5 cm with a non-stretching tape placed

around the abdomen at the iliac crest. Body fat percentage was assessed by a validated bioimpedance analysis device (Omron®BF306, Omron Healthcare Europe BV, Hoofddorp, The Netherlands) [6]. After entering demographic and anthropometric data, the subjects were asked to remain in standing position while holding the hand-to-hand bioimpedance device by both hands and straightening both arms forward [7]. All demographics, anthropometric and lifestyle information were prospectively collected by a trained nurse.

An Abbott chemiluminescent microparticle immunoassay, which measures both 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3, was applied for the determination of total 25-hydroxy vitamin D concentration in serum [8]. At an average total concentration of 49 nmol/L, 99 nmol/L and 187 nmol/L, the intra-assay coefficient of variation was 3.9%, 4.0%, and 4.0%, respectively. The corresponding inter-assay coefficient was 1.0%, 1.2%, and 2.6% [8]. Accuracy and reliability of the assay are assessed both in the Vitamin D Standardization Program [9] and in the Vitamin D External Quality Assessment Scheme [10]. The circulating levels of total cholesterol (enzyme assay) and ferritin (immunoassay) were measured in serum. All laboratory assessments were performed in the same accredited central laboratory (Viollier, Basel, Switzerland) using an Architect CI8200 (Abbott, Chicago, IL, USA) analyzer. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Southern Switzerland (RIF CE 2775). Informed written consent was obtained from all subjects to participate in the study.

#### *Data Analysis*

Frequency distribution of continuous data were presented as median and interquartile range. Dichotomous data were presented as absolute and relative frequency. Concentrations of total 25-hydroxy-vitamin D were considered adequate if ≥50 nmol/L–≤250 nmol/L, insufficient if <50 nmol/L, deficient if <30 nmol/L or potentially toxic if >250 nmol/L [11]. Anthropometric, lifestyle and further laboratory characteristics of the subjects with adequate (50–250 nmol/L) and insufficient (<50 nmol/L) 25-hydroxy-vitamin D values were compared by Kruskal-Wallis test. χ<sup>2</sup> test was used for comparing frequencies of categorical variables. The Bonferroni test adjustment for multiple comparisons was applied.

Odds ratios (ORs) of 25-hydroxy-vitamin D insufficiency and corresponding 95% confidence intervals (CI) from univariate logistic regression models were calculated for the following variables: age, body height, body weight, body mass index, body fat percentage, frequency/length and site of recreational physical activity, frequency of alcohol consumption, smoking, season (winter from 21 December; spring from 21 March, summer from 21 June and autumn from 21 September), cholesterol and ferritin concentrations. ORs of 25-hydroxy-vitamin D insufficiency and corresponding 95% CI were also derived from the best AIC-selected multiple logistic regression model including the following variables: age, body mass index, body fat percentage, waist circumference frequency/length and site of recreational physical activity, frequency of alcohol consumption, smoking, season, cholesterol and ferritin concentrations. In all analyses, significance was assumed if *p* < 0.05. Statistics was performed using the open source statistical language R, Vienna, version 3.5.3 (11 March, 2019).

#### **3. Results**

Body height (178.0 (173.5–182.0) vs. 177.5 (173.0–182.5) cm) and weight (72.2 (65.7–80.0) vs. 72.0 (65.0–80.5) kg) were similar in subjects who volunteered to participate in the study as compared with the remining 3618 subjects. Anthropometric, lifestyle and laboratory findings of the 1045 recruited subjects are given in Table 1. One hundred seventy-nine (17%) subjects presented with concentrations of total 25-hydroxy-vitamin D < 50 nmol/L. Among subjects with a concentration of vitamin D below 50 nmol/L, 24 (13%) had deficient levels of total 25-hydroxy-vitamin D. No subject presented with potentially toxic concentrations of the 25-hydroxy-vitamin D. The concentration of 25-hydroxy-vitamin D2 was always ≤5 nmol/L. The characteristics of the subjects with adequate or insufficient concentrations

of 25-hydroxy-vitamin D are shown in Table 2. A total of 76 (7.2%) out of 1045 had a body mass index <sup>≥</sup> 30 kg/m<sup>2</sup> and 34 (3.3%) <sup>≤</sup> 18.5 kg/m2.

The season of measurement was the most significant factor associated with insufficient concentrations of 25-hydroxy-vitamin D. The concentrations vitamin D in the four seasons are depicted in Figure 1 (upper panel). Of note, 64 (38%) out of 170 subjects tested for 25-hydroxy-vitamin D level in winter presented insufficient concentrations of this vitamin, 70 (18%) out of 383 in spring, 18 (5.4%) out 331 in summer and 28 (17%) out of 161 in autumn. A total of 13 (7.6%) subjects in winter, 6 (1.6%) in spring and 5 (3.1%) in autumn, presented with deficient concentrations of 25-hydroxy-vitamin D. No subject had a deficient level of 25-hydroxy-vitamin D in summer (Figure 1, lower panel).


**Table 1.** Baseline characteristics of the enrolled subjects. Data are given as absolute frequency (and percentage) or median (and interquartile range).

**Table 2.** Characteristics of subjects with adequate and insufficient circulating 25-hydroxy-vitamin D. All variables were non-normally distributed. Data are given as absolute frequency (and percentage) or median (and interquartile range). The Kruskal-Wallis test was used for continuous variables. Chi-squared test was used for categorical variables. The Bonferroni test adjustment was applied to account for multiple comparisons.


\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001.

**Figure 1. Upper panel.** Box-plot of the circulating concentrations of 25-hydroxy-vitamin D across the four seasons. The boxes include values between the 1st and the 3rd quartile (i.e., the interquartile range). The whiskers include values between: 1st quartile − 1.5 times interquartile range and 3rd quartile + 1.5 times interquartile range. **Lower panel.** Frequency of adequate, insufficient or deficient concentrations of 25-hydroxy-vitamin D according to seasonality.

In the univariate logistic regression models (Table 3), body height (ORs 0.96, 95% CI 0.94–0.98), body mass index (OR 1.05, 95% CI 1.00–1.07, body fat percentage (OR 1.04, 95% CI 1.02–1.07), waist circumference (OR 1.02, 95% CI 1.00–1.03), the frequency of recreational physical activity 5–6 per week (OR 0.36, 95% CI 0.16–0.85), cigarettes consumption of 11–20 cigarettes per day (OR 0.41, 95% CI 0.22–0.77), the season (spring, OR 0.37, 95% CI 0.25–0.56, summer, OR 0.09, 95% CI 0.05–0.17, and autumn, OR 0.33, 95% CI 0.20–0.56) and cholesterol (OR 1.29, 95% CI 1.02–1.62) were positively (OR >1) or inversely (OR < 1) associated with the risk of 25-hydroxy-vitamin D insufficiency.

Table 4 shows results from the multiple regression analysis. After model selection based on clinical plausibility and Akaike information criterion, the increase of body fat percentage was a significant risk factor (ORs >1) for 25-hydroxy-vitamin D insufficiency. A frequent (5–6 times per week) and outdoor physical activity and non-winter seasons were significant protective factors (ORs < 1) against 25-hydroxy-vitamin D insufficiency.


**Table 3.** Odds ratios (ORs) of 25-hydroxy-vitamin D insufficiency and corresponding 95% confidence intervals (CIs) from univariate logistic regression models.

\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.0001.

**Table 4.** Odds ratios (ORs) of 25-hydroxy-vitamin D insufficiency and corresponding 95% confidence intervals (CIs) as derived from the best Akaike information criterion-selected multiple logistic regression model. The original model included the following variables: body mass index, body fat percentage, mid-upper arm circumference, waist circumference, frequency of recreational physical activity, duration of recreational physical activity session, site of recreational physical activity, season, frequency of alcohol consumption, smoking, cholesterol and ferritin.


\* *p* < 0.05, \*\*\* *p* < 0.0001.

#### **4. Discussion**

This study points out that a large minority (17%) of healthy male late adolescents in Southern Switzerland, a region with a low natural vitamin D intake, has a poor vitamin D status. In this group of subjects, an increase in body fat percentage is a risk factor for vitamin D insufficiency. On the contrary, frequent and outdoor recreational physical activity and, especially, non-winter seasons are protective factors against vitamin D insufficiency. Yet, about one every fifth subject has insufficient concentrations of vitamin D also in spring and autumn.

The prevalence of hypovitaminosis D among adolescents from high-income countries largely varies among studies [12,13]. In the southeastern United States, vitamin D concentrations < 50 nmol/L were observed in about 4% of white male adolescents [14]. In the HELENA study, among 1006 subjects living in ten European countries, about 40% presented vitamin D concentration < 50 nmol/L [15]. Our data point out that insufficient concentrations of vitamin D are frequent among male late adolescents in Southern Switzerland and emphasize that vitamin D concentrations are strongly season dependent. Of note, the frequency of vitamin D insufficiency was very high in winter and still rather important (>15%) in autumn. A previous study suggests that in Ireland (latitude 51–55◦) ultraviolet B radiation is effective for some vitamin D synthesis also in October [16]. On the other hand, very low doses were found in November and December. This study did not specifically investigate the ultraviolet B radiation in Southern Switzerland. However, the concentration of 25-hydroxy-vitamin D2 was always ≤5 nmol/L, confirming that also vitamin D of non-animal origin played a marginal role in vitamin D status in our population.

The peak bone mass is usually reached between 25 and 35 years of age and, in male subjects, is predicted by vitamin D status [17]. Hence, the years preceding the peak bone mass are considered as a critical period to maximize bone strength and maturation [18]. The European Academy of Pediatrics, the American Academy of Pediatrics and Endocrine guidelines currently do not routinely recommend supplying vitamin D in non-dark skinned, non-obese healthy adolescents or young adults [19–21]. The results of this study suggest that longitudinal studies should address the advantages of vitamin D

supplementation in Caucasian late adolescents during winter. This finding is even more crucial for the bone metabolism, considering that only vitamins D concentrations >75 nmol/L have a clear-cut antifracture effect [22]. On the other hand, an increasing body of evidence highlights that vitamin D deficiency is associated with chronic and potentially life-threating conditions such as cardiovascular disease later in old adults and elderly [23]. In this study, about one every thirteen subjects had deficient concentrations of vitamin D in winter.

We found an association between vitamin D concentrations, frequent and outdoor physical recreational activity after adjusting for confounders. Although sun-light exposure does not occur exclusively during recreational physical activity, this finding confirms the beneficial role of outdoor activities. Yet, seasonal fluctuations of ultraviolet-B radiations might decrease the effects of sun-light exposure during non-summer seasons and especially in winter [22,23]. Differently from previous observations [24,25], this study did not identify any association between vitamin D and a marker of inflammation such as ferritin. This might be due to the fact that the population of this study exclusively included healthy late adolescents without any chronic disease. Also, we did not find any association with cholesterol or alcohol consumption. A possible explanation is that most prior studies have included subjects with a much broader range of age and the mentioned factors could become determinant when persisting for long-term periods [26]. Previous studies found body mass index to be inversely associated with vitamin D concentrations. However, residual confounding such as physical activity or body composition assessment have not always been considered [27]. Furthermore, body mass index cannot distinguish lean from fat mass, especially in youth [28]. One of the advantages of this study is that many anthropometric characteristics were explored disclosing an association between body fat percentage and vitamin D concentrations levels after adjusting for confounders. A tendency to accumulate vitamin D (a liposoluble compound) in fat depots or an impaired vitamin D intestinal absorption or hydroxylation in adipose tissue are likely to underly this association [29]. Of note, some studies have also hypothesized that vitamin D insufficiency itself could reduce weight loss or favoring weight gain [29].

This study has many strengths and limitations. The main strengths are the large number of apparently healthy subjects enrolled with a narrow range of age and the concomitant determination of many potential modulators of vitamin D including the body fat percentage. Furthermore, Southern Switzerland is considered the sunniest region of Switzerland: therefore, it is possible that the number of late adolescents with vitamin D insufficiency might be even higher in the other parts of Switzerland. The main limitation of this study is the exclusion of females. Second, results are partly based on self-reports, which might not always correspond to the actual behavior of the responders. Third, its cross-sectional nature prevents to longitudinally evaluate vitamin D concentrations throughout the seasons. Fourth, we did not analyze some common inflammatory indices, such as C-reactive protein. Finally, the use of sunscreen, which is usually not very common among male adolescents and young adults in Switzerland [30], was not investigated.

#### **5. Conclusions**

This study showed that about one every fourth healthy male late adolescent in Southern Switzerland presents insufficient concentrations of vitamin D during non-summer seasons. Low body fat and both frequent and outdoor recreational physical activity are associated with adequate vitamin D level this population.

**Author Contributions:** Conceptualization, F.M., M.G.B., A.S. and G.S.; Methodology, F.M., A.R., S.A.G.L., V.E. and G.P.M.; Formal Analysis, V.E. and G.P.M.; Investigation, F.M., A.S., S.A.G.L. and M.G.B.; Data Curation, A.R., G.P.M. and G.S. Writing—Original Draft Preparation, G.S., M.G.B., F.M. G.P.M.; Writing—Review & Editing, A.R., S.A.G.L., V.E., A.S.; Supervision, G.S.; Project Administration, F.M.; Funding Acquisition, F.M.

**Funding:** The study was supported by the Swiss Society of Hypertension.

**Acknowledgments:** Authors thank Silvia Muggli for data check.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **E**ff**ects of Vitamin D Status and Supplements on Anthropometric and Biochemical Indices in a Clinical Setting: A Retrospective Study**

#### **Myriam Abboud 1,2, Xiaoying Liu 1, Flavia Fayet-Moore 3, Kaye E. Brock 1, Dimitrios Papandreou 2, Tara C. Brennan-Speranza <sup>1</sup> and Rebecca S. Mason 1,\***


Received: 18 November 2019; Accepted: 10 December 2019; Published: 12 December 2019

**Abstract:** Context: Obesity and low vitamin D status are linked. It is not clear that weight loss through lifestyle intervention is influenced by vitamin D status. Objective: The aim of this study was to investigate the effect of baseline vitamin D status and vitamin D supplementation on weight loss and associated parameters for participants on a weight loss program in a primary care setting. Design: A retrospective analysis of clinical records of patients who underwent an individually tailored weight loss program at a single dietetic clinic in Sydney, Australia. Setting: Primary care centers. Patients: 205 overweight and obese men and women aged from 18 to 50 years. Interventions: Patients were referred to a dietetic clinic for a weight loss program. Patients with low serum 25-hydroxyvitamin D (25(OH)D) concentrations at baseline were advised to increase sun exposure and take multivitamins supplemented with 2000 IU or 4000 IU per day of vitamin D3, according to the preference of their primary care physician. Main outcome measures: Clinical parameters of weight, height, waist circumference, and serum 25(OH)D, as well as blood pressure and fasting lipid profile were collected from both baseline and three-month follow-up consultations. Results: Subjects with sufficient baseline 25(OH)D levels (≥50 nmol/L) experienced significantly greater weight loss (−7.7 <sup>±</sup> 5.9 kg vs. <sup>−</sup>4.2 <sup>±</sup> 3.3 kg) and reductions in BMI (−2.6 <sup>±</sup> 1.8 kg/m2 vs. <sup>−</sup>1.5 <sup>±</sup> 1.1 kg/m2) and waist circumference (−5.2 ± 3.5 cm vs. −3.1 ± 3.1 cm) as compared with those who were vitamin D insufficient at baseline (*p* < 0.001 for all). Vitamin D insufficient patients who were supplemented with daily 2000 IU or 4000 IU vitamin D experienced significantly greater decreases in weight (−5.3 <sup>±</sup> 3.6 kg vs. <sup>−</sup>2.3 <sup>±</sup> 1.6 kg), BMI (−1.9 <sup>±</sup> 1.2 kg/m2 vs. <sup>−</sup>0.8 <sup>±</sup> 0.6 kg/m2) and waist circumference (−4.2 ± 3.4 cm vs. −1.2 ± 1.3 cm) as compared with those not supplemented (*p* < 0.001 for all). We also observed a greater decrease in low-density lipoprotein (LDL) cholesterol (−0.4 ± 0.5 mmol/L vs. −0.2 ± 0.5 mmol/L) in subjects insufficient at baseline and supplemented as compared with those insufficient at baseline and not supplemented (*p* < 0.01). Conclusion: In a weight loss setting in a dietetic clinic, adequate vitamin D status at baseline, or achieved at three months through supplementation, was associated with significantly greater improvement of anthropometric measures. The study has implications for the management of vitamin D status in obese or overweight patients undergoing weight loss programs.

**Keywords:** vitamin D deficiency; 25-hydroxyvitamin D; vitamin D supplements; weight loss; low-density lipoprotein (LDL) cholesterol; high-density lipoprotein (HDL) cholesterol; triglycerides (TG); blood pressure

#### **1. Introduction**

Vitamin D has multiple pleiotropic functions beyond its traditional role in calcium homeostasis, as well as bone and muscle function [1]. Actions of the vitamin D hormone, calcitriol, have been demonstrated in many tissues, including adipocytes [2] and the cardiovascular system [3]. The first evidence of a relationship between vitamin D and body fat was described in 1972 by Mawer et al. [4]. Inadequate vitamin D status, obesity, and chronic noncommunicable disease often cluster [1,3,5–7]. They are important public health issues that contribute significantly to modern healthcare costs, morbidity, and mortality [8,9]. Many studies support the proposal that obesity could be driving low serum 25(OH)D concentrations mainly due to decreased bioavailability of vitamin D through sequestration in body fat compartments [4,10–13]. There is limited human research which indicates that vitamin D could potentiate weight loss and improvements in metabolic markers [14,15]. A recent randomized controlled trial in postmenopausal women reported that while supplementation with vitamin D did not alter weight loss or associated parameters overall as compared with a placebo group, women in the supplemented group who reached 25(OH)D concentrations of ≥32 ng/mL (≥80 nmol/L), had greater improvement in several measured weight loss parameters as compared with those women whose final 25(OH)D concentrations were below 80 nmol/L [16].

In this study, we hypothesized that overweight and obese patients presenting adequate 25(OH)D levels will have a greater reduction in body weight, body mass index (BMI), and waist circumference as compared with those with inadequate vitamin D levels while undergoing a three-month clinic-specific individually tailored weight loss management program. Furthermore, we expect that vitamin D repletion of those who were insufficient at baseline, through short-term daily vitamin D supplementation would enhance weight loss, decrease waist circumference, and improve biochemical markers. This was investigated using clinic records of a population of overweight and obese men and premenopausal women who participated in an individually tailored three month weight loss program.

#### **2. Study Design and Population**

This study is a retrospective analysis of a clinical databank that was recorded in a health giver-receiver setting. The Human Ethics Committee at the University of Sydney approved the research protocol (Protocol 2013/206). Between September 2011 and March 2013, a total of 935 patients who attended three medical centers in Sydney, Australia, were referred to a dietetic clinic (established by author MA) to assist with a program for weight loss, under the Chronic Disease Management Plan of Medicare Australia [17]. This care plan entitled each patient to five consultations with a dietician or another allied health professional. Under an agreed protocol, patients had blood taken for 25(OH)D and blood lipid measurements at the initial visit with the primary care physician. These patients were seen by the dietician fortnightly for the first month and then monthly after that. Thus, the initial visit was the first visit, then, after two weeks (second visit), then, another two weeks (third visit), then, after one month (fourth visit), then, after one month (fifth visit). This fifth visit coincided with the three-month follow-up, when the follow-up blood for testing was taken. The referring doctors differed in their approach to management of patients who had 25(OH)D concentrations less than 50 nmol/L at baseline. Some referring doctors advised their patients to take supplements of vitamin D3 at 2000 IU per day, and some at 4000 IU per day. The remaining patients with low baseline 25OHD were advised to increase their sun exposure and take multivitamins (with only small amounts, 40 IU of vitamin D). The dietician performed anthropometric measures at each visit. Weight was measured using the same scales, which were calibrated monthly. Height and waist circumference were measured at the

initial and final visits. Waist circumference was measured with the patient standing, at a level midway between the iliac tubercle and lower lateral rib margin, and hip circumference was measured at the level of the iliac tubercle.

For the individually tailored weight loss protocol, at the initial consultation with the dietician, each patient's daily estimated energy requirement (EER) was calculated using the Harris-Benedict equation [18] and physical activity factors (see more detailed information in the supplementary Tables S1–S3). Overweight and obese individual caloric goals were calculated to be 300 and 500 Kcal/day, respectively, less than their EER. Once the EER was calculated, a meal plan was designed by the dietitian and given to the participant at the initial consultation, and adherence was checked via a 24-h recall method during each of the follow-up visits. The reported intake was relatively compliant with the prescribed energy intake. The participants were not seen by any exercise physiologist and did not undertake any major changes in physical activity that could have altered their energy needs.

As part of continuing care, a report was sent to the referring primary care physician requesting a follow-up on 25(OH)D and other biochemical markers at three months from the initial consultation, which coincided with the final dietary consultation. This blood test was performed on the same day as the final dietary consultation.

All 935 records of patients who were referred to this program between September 2011 and March 2013 were examined. Of these, 676 records were excluded based on the following predetermined exclusion criteria: a history of diabetes mellitus, polycystic ovary syndrome, parathyroid disorder, kidney or liver disease, osteopenia or osteoporosis, or current pregnancy, or taking any medication known to affect body weight (such as steroids) or supplements such as calcium or vitamin D (>400 IU of vitamin D2 or vitamin D3, not prescribed as part of this intervention). A further 47 patients were excluded as they did not complete the follow-up blood test at three months. There were seven subjects in the group which had sufficient 25(OH)D concentrations (≥50 nmol/L) at baseline, who received vitamin D supplements. These were also excluded from the analysis.

Records of 205 healthy men and premenopausal women between the ages of 18 and 50 were coded for analysis. Clinical parameters including blood pressure; fasting lipid profile, i.e., total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TG), and serum 25 hydroxyvitamin D (25(OH)D; as well as anthropometric measurements (weight, height, and waist circumference) were collected from both baseline and three-month follow-up consultations. Patients reported sun exposure frequency at baseline and exercise levels.

Overweight and obesity were classified according to BMI (overweight 25 to 29.9 kg/m<sup>2</sup> and obesity <sup>≥</sup>30 kg/m2) and waist circumference (overweight men 94.0 to 101.9 cm, women 80.0 to 87.9 cm, obesity men ≥102.0 cm, and women ≥88.0 cm).

#### *Biochemistry*

Plasma levels of cholesterols and triglycerides were determined by standard laboratory methods and were all performed by Laverty Laboratory, North Ryde, Sydney, Australia. They were measured using an enzyme-based Siemens platform, where LDL was calculated in accordance with the Friedewald equation (18). Normal ranges for lipid profile were provided by the commercial laboratory: TC (3.5–5.4 mmol/L), LDL (2.1–4 mmo/L), HDL (>=1 mmol/L), TG (0.1–2 mmol/L). Plasma 25(OH)D concentrations were all determined at the Laverty Laboratory using the Diasorin Siemens chemiluminescent assay and vitamin D insufficiency was defined as 25(OH)D level <50 nmol/L [19]. The assay characteristics are described in [20].

#### **3. Statistical Analysis**

All analyses were performed using SPSS for Windows (version 17.0 SPSS, Inc., Chicago, IL, USA). Analyzed data were collected from a clinical setting with intention-to-treat approach. Differences in anthropometric and blood parameters between patients who had sufficient baseline 25(OH)D, and insufficient baseline 25(OH)D with or without prescription of vitamin D supplementation, were

assessed by one-way ANOVA followed by Tukey's post-test. Comparisons for the within-group changes in Table 1 were made using paired Student t-tests. Correlations were assessed by calculating Pearson correlation coefficients. LOESS plots [21] were calculated by the SPSS program. Multivariate analysis of changes in weight, BMI, and waist circumference were regressed against 25(OH)D concentrations at follow-up using stepwise linear regression models using the following independent variables: 25(OH)D values at follow-up, adjusting for age, sex, season of baseline appointment, sun exposure, and exercise and were split by prescription of vitamin D supplements. In the initial analyses, the subjects who were vitamin D insufficient at baseline (25(OH)D <50 nmol/L) and supplemented with 2000 IU vitamin D3 per day, were analyzed separately from those who were supplemented with 4000 IU/day. There were no differences between these groups in terms of baseline parameters, except for baseline 25(OH)D which was significantly lower at 31 ± 13 nmol/L in the subjects who were prescribed 4000 IU per day, as compared with 39 ± 16 nmol/L in those prescribed 2000 IU per day (*p* < 0.02). For ease of data presentation and statistical power, these supplemented groups have been combined.



25(OH)D indicates serum 25-hydroxyvitamin D; HDL, high-density lipoprotein; LDL, low-density lipoprotein; and BP, blood pressure. Data are shown as mean values ± SD data from 205 subjects, except for total cholesterol and triglycerides (204), LDL (194), and HDL (197). *p* values show differences between baseline and follow-up values, NS, non-significant *p* > 0.05.

#### **4. Results**

#### *4.1. Subject Characteristics*

As shown in Table 1, there was a significant overall reduction in weight, BMI, waist circumference, systolic blood pressure, LDL, and triglycerides after the three-month weight loss program.

There were 70 men and 135 women whose records were included in the study. Although the men were significantly older (mean 39 years vs. 37 years, *p* = 0.007) and had significantly higher weight, waist circumference, blood pressure, LDL, and triglyceride values, and lower HDL, at baseline, they were not significantly different from the women in terms of baseline BMI, 25(OH)D, or the proportion who were vitamin D sufficient (see Supplementary Table S1). At three months, there were again no significant differences between males and females in terms of BMI, 25(OH)D concentrations, or the proportion who were vitamin D sufficient, but differences in the other parameters persisted (Supplementary Table S2). Sex had no significant effect on changes in weight, BMI, waist circumference, 25(OH)D concentration, or total cholesterol over the three-month period of analysis (*p* values of >0.05 for all). For this reason, in Table 1 and subsequent tables, data for male and female subjects have been combined.

Consistent with the high prevalence of vitamin D insufficiency with obesity, the mean baseline serum 25(OH)D concentration was insufficient (45 ± 19 nmol/L) and the mean baseline BMI classified subjects as obese overall. At baseline, 3% of the baseline subjects were in the normal weight range, 50% were overweight, and 47% were obese. After three months on a weight loss program and

supplementation with vitamin D for some individuals, the mean serum 25(OH)D level was significantly higher than that of the baseline at 54 ± 17 nmol/L (*p* < 0.001) with a three month median and interquartile range of 55 and 23 nmol/L, respectively, while the mean BMI and waist circumference were significantly lower than that of the baseline (Table 1). After three months on the program, 22% of subjects were normal weight, 45% were overweight, and 33% were obese.

#### *4.2. E*ff*ect of Vitamin D Status and Supplementation on Anthropomorphic Measures and Other Parameters*

Baseline serum 25(OH)D was significantly higher in the sufficient group as compared with the deficient group (64 ± 11 vs. 33 ± 10 nmol/L, *p* < 0.001, Table 2). After three months on the program, 25(OH)D concentrations were similar to baseline values in both vitamin D sufficient and vitamin D insufficient individuals not given supplemental vitamin D, despite advice to increase sun exposure and take multivitamins (Table 2).

**Table 2.** Baseline values for anthropomorphic measures, lipids, and blood pressure, and baseline and follow-up values for 25(OH)D with or without the three month supplementation with vitamin D. Values are presented as means ± SDs.

