**Abstract**

How happiness is defined depends on who is asked. In the case of universities, student happiness should prevail, yet their voices are often overlooked. This is also the case in the research literature where non-Western views are less frequently reflected. The United Arab Emirates (UAE), a country whose population is comprised of nearly 90% expatriate residents, is a good study case as campuses are filled with international students and the wellbeing of residents is a national priority. Responses from 80 UAE-based expatriate students reveal they are happiest with friends and in social activities and interactions; they want more opportunities to connect socially in classrooms and campuses, efforts which can be crafted by faculty. They also want joyful, inspiring learning where they can discuss and be exposed to other views, with many driven by the need to feel productive, efficacious and engaged. Solutions included the need for faculty to develop warmer student relationships, the maintenance of online learning to reflect realities of work and relationships, and for students to be treated with more respect as fee-paying adults. As the number of international students rise, research into their happiness and what universities can do to increase it remains a global priority.

**Keywords:** happiness, university, institutions, students, qualitative, belonging, social connections

### **1. Introduction**

University is a time of transition; greater independence and less oversight mark the social, learning, and emotional worlds of young adults. With freedom to make life choices, have fun, and choose one's friends, there is also more isolation and stress, financial worry, and learning struggles [1–3]. Early adulthood's slope is slippery; many young people thrive, but some flounder, and others even suffer. Indeed, mental health is taxed upon university entrance with depression, anxiety, and stress peaking around the age of 25 [4–6]. In the Middle East/North Africa region, depression and

anxiety in young people are higher than global averages [7, 8] and the prevalence of mental health issues is growing with implications for learning, quality of life, relationships and future work outcomes.

Yet, greater than the mere absence of illbeing, the presence of wellbeing is also conducive to a good life. Young people who experience greater life satisfaction and more frequent experiences of positive emotion are more likely to attain a postsecondary degree, search for work, be hired and promoted, as well as get a higher salary [9–11]. Studies conducted in schools and universities show that greater wellbeing boosts learning by as much as 6 months of additional learning and reduces the chances of poor performance [12–15]. Greater wellbeing also spells fewer mental health issues such as depression, loneliness, and anxiety into adulthood [16, 17]. More than simply "nice to have", wellbeing is protective as well as promotive of good living, social relating and learning.

Still, wellbeing is often positioned as the responsibility of counseling departments alone and often takes the form of consumable skills and awareness raising initiatives [1, 3, 18]. By construing the psychosocial needs of students as individual mental health issues, institutions avoid the responsibility of building positive ecosystems. Yet, with local stigma around help-seeking and poorly resourced mental health services [19, 20], alongside post-COVID learning losses and weak performance on university exams more generally [21, 22], and ongoing future economic and employment uncertainty, learning institutions must and can do more [2, 3, 23–25]. Further, as many countries are now focused on wellbeing as a national priority, as is the case in the United Arab Emirates (UAE) where this study is situated, educational institutions have additional incentive to meet this national aim, although universities have been slow to adopt the view that wellbeing matters. Knowing what students perceive as helpful is necessary. Comprised of nearly 90% expatriates, the UAE is a hub for university students. Soliciting their views can help pave the way towards developing more responsive institutions.

### **1.1 "Positive universities": more than learning and profit**

Positive universities [26] have recently been identified in the field of positive psychology as institutional ecosystems that are expressly designed to nurture and facilitate student wellbeing. Positive universities explicitly prioritize wellbeing as much as academic performance. To drive wellbeing agendas, they have at their disposal a range of drivers including potential curriculum adjustments, special for-credit wellbeing courses, campus initiatives, architectural design specifications, wellbeing policies aimed at student, administrative, learning and management processes, as well as aims to strengthen interpersonal relationships. Topics such as fairness, inclusion and integrity, as much as joy, curiosity, depression and loneliness are routinely discussed. This focus is primarily aimed at students, but the wellbeing of faculty, employees, as well as leadership and management is also maximized. Wellbeing is construed as a business imperative given its links to organizational productivity, turnover, profit, and customer loyalty [27–29]. It is equally a learning instrument and student retention tool given that it is tied to employability, university drop-out rates, academic learning and future work satisfaction [30, 31]. Positive institutions are preventive as much as proactive in nature, predicting what students, faculty, management, employers, and community stakeholders need to emotionally thrive. Parents, alumni, and institutional partners are also actively involved. Positive universities deliver students ready for work and more importantly, prepared to contribute to society and maximize their own potential.

### *Thanks for Asking! How UAE-Based University Students Conceptualize Happiness and How… DOI: http://dx.doi.org/10.5772/intechopen.107363*

Yet, the reality is that being a university student is often marked by loneliness, lack of meaning, and low or negative moods [1–3]. For students in international forms of education, there is often a disconnect between student's prior education systems and that which is being offered, such that independent critical thinking, student-led motivation, academic writing, defending one's ideas, and group-based learning conflicts with memorization and rote learning [32–34]. English language proficiency not only affects learning, but socialization and the ability and willingness to ask for assistance [32, 34, 35]. That nearly all faculty are also expatriates and have their own culturally embedded notions of learning and relating is another adjustment students make [34].

Further, the wellbeing of young people has often been overlooked as institutions do not consider it their concern [36]. In the UAE, young people are often "Third Culture Kids"; that is, not a product of their parent's home country (i.e., in some cases parents come from two separate countries, in which the young person has spent no time), and not belonging to the UAE national culture either, making needs for attachment and belonging salient. Further, after completing secondary school, they transition to higher education while continuing to live under their parent's roof and are often more motivated to spend time on campus in a bid for independence [37]. Their continued parental living arrangements and expatriate status has paradoxically meant institutions construe wellbeing as a private family and not an institutional concern.

Moreover, in the Arabian Gulf emerging economies, the focus of private institutions as new operating campuses is to prosper in competitive institutional markets, climb international rankings and generate profit for local investors or home campuses [34, 38–40]. Efforts to stem costs have meant that community service, professional development, wellbeing and mental health commitments are not priorities. With the number of international students rising and the internationalization of higher education growing globally, including in the Gulf [34, 38, 39], institutions have an interest in developing themselves to meet the demand alongside the psychosocial and academic needs of those who attend them.

### **1.2 The case for asking what matters**

Despite growing interest in wellbeing, there is little consensus around what it means [41–43], especially at university [3]. It is often used interchangeably with the umbrella term of "happiness," referring more generally to an affective state of feeling good, while "wellbeing" refers to, and also includes functioning well over time. Further, while research to guide initiatives grows, what is relied upon more often stems from the West (e.g., [1]) and is based on "WEIRD" samples, i.e., Western, Educated, Industrialized, Rich, Democratic [44] that is not always helpful elsewhere. Further, the most recent iteration of positive psychology 3.0 construes wellbeing from a systems perspective, whereby contexts are the unit of intervention and understanding, versus individuals alone [41, 45]. This removes the onus on individuals to singularly engage with their wellbeing, instead sharing that responsibility with the systems in which they operate.

There is also a growing importance of the need to include participant voices, often via a qualitative lens to produce insights that are not available through standardized means, but also to understand how wellbeing is lived [46, 47]. The lay conceptions that individuals hold, that is, their personal beliefs about the nature of their own happiness, its values, antecedents, as well as outcomes, highlight what matters to them and reflect their values and experiences [48–50]. Listening to the views of young people as consumers of education and wellbeing can help institutions guide actions, policies, and initiatives accordingly [2, 51].

Indeed, research involving young people often reveals a disconnect between the approaches they deem important versus those put forward by institutions [52]. For example, in school, young people identified the need for informal, immersive ways to foster social connectedness and support, over individual skills-based tools and strategies [18, 52]. Similar results have been found in a group of Chinese university students studying in Australia who identified wellbeing offerings they felt would help them best, including cross-cultural interactions, non-discriminatory campus environments and greater support with employment and housing opportunities [48]. These were in great contrast with what was being offered on campus, such as stress-management services and individual counseling. Considering students as both consumers and coproducers of interventions [53] alongside other institutional and community changes can give them voice, a wellbeing intervention itself, and remove the burden from either group solving wellbeing challenges alone.

In sum, higher education has a role in strengthening student wellbeing [1–3, 23, 48]; yet individual-level interventions designed to "fix" students rather than identifying the institutional drivers that fuel problems are often preferred. Inquiry into the broader systemic factors to support the wellbeing of young people is growing as institutional transformations, like physical space, part-time study options, faculty training in wellbeing, and policies around mental health, diversity and inclusion for example, may have larger impacts than individual intervention [1, 3, 54]. Thus, what institutions can and should do is growing as a topic of scientific inquiry, as well as a business imperative. Accordingly, we explore these views.


Differences were observed in mean S-25(OH)D levels between all ethnic groups compared with the white grouping (mean 64.48 nmol/L), except for the West Asian and Korean groups (Table 3). The lowest mean was reported for the Japanese (33.16 nmol/L; *p* < 0.001), followed by Arabs (37.14 nmol/L; *p* < 0.001), and Southeast Asians (40.92 nmol/L; *p* < 0.001). Compared with the white grouping (6.61%), more than half (55%) of the Japanese ethnic group had vitD deficiency, with similar proportions found for Arabs (52%) and Southeast Asians (41.1%). Moreover, the Japanese had the highest insufficiency level (85%), followed by Koreans (81.0%), and Southeast Asians (73.8%). The ranges for insufficiency (30–49 nmol/L) and sufficiency (50–74 nmol/L) for each ethnic group are also presented in Figure A1, Appendix B.

Table 4 highlights the marked differences among those born in Africa, Asia, and South/Central America, and Caribbean regions. The highest rates of deficient and insufficient vitD were found among immigrants from Africa (31.2% and 68.6%, respectively) and Asia (24.5% and 69.7%, respectively) compared with those born in Canada and North America (7.7% and 31%, respectively). The ranges for insufficiency (30–49 nmol/L) and sufficiency (50–74 nmol/L) for the above mentioned five regions are presented in Figure A2, Appendix B.

**Table 3.** Weighted mean S-25(OH)D (nmol/L) and vitamin D status by ethnicity using Cycles 3 and 4 of Canadian Health Measures Survey data.

Note: following the Statistics Canada guideline, some cells merged due to the low number of participants. † Reference value; SE, standard error; CI, confidence interval. *p*-values: \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001.

> The highest mean S-25(OH)D level was found in those born in the Netherlands, followed by the UK and Germany (72.7, 68.9, and 68.5 nmol/L, respectively). The lowest mean levels of S-25(OH)D were found in those from Algeria (34.7 nmol/L, *p* < 0.001), Lebanon (39.4 nmol/L, *p* = 0.008), and Morocco (39.4 nmol/L, *p* = 0.003). The highest deficient levels were found in those from Morocco (55.9%), India (34.4%), and Lebanon (about 30%), with the highest insufficient levels in those from Algeria (87.9%), Romania

(80.7%), and Lebanon (78.7%). The ranges for insufficiency (30–49 nmol/L) and sufficiency (50–74 nmol/L) for each birth country are presented in Figure A3, Appendix B.

**Table 4.** Weighted mean S-25(OH)D (nmol/L) and vitamin D status by geographical region and country of birth using Cycles 3 and 4 of Canadian Health Measures Survey data.

Note: following the Statistics Canada guideline, some cells merged due to the low number of participants. † Reference value; SE, standard error; CI, confidence interval. *p*-values: \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001.

#### *3.4. S-25(OH)D Concentration and VitD Status by Immigration Status and Season*

The S-25(OH)D increased substantially during summer and fall compared with winter. Compared with January, the lowest weighted mean was found in February and the highest in September. The highest percentage of change (−37%) from the optimal level (75 nmol/L) was in February (Figure 1) and (Table A5, Appendix B). Non-immigrants had higher S-25(OH)D in all seasons, with significant increments in spring, summer, and fall compared with the winter season (Figure 2) and (Table A6, Appendix B).

**Figure 1.** Weighted mean S-25(OH)D (nmol/L)/month of test and percentage of change from the optimal level (75 nmol/L) using Cycles 3 and 4 of Canadian Health Measures Survey data († reference value \* significant at *p* ≤ 0.05).

**Figure 2.** Weighted seasonal variation in mean S-25(OH)D (nmol/L) based on immigration status using Cycles 3 and 4 of Canadian Health Measures Survey data († reference value \* significant at *p* ≤ 0.05).

#### *3.5. Results of Multivariate Analysis*

The final multi-linear regression model showed that immigrants had lower S-25(OH)D levels compared with non-immigrants (beta-estimate: −5.28, 95% CI: −7.48, −3.09, *p* < 0.001) after adjusting for all covariates (Table 5). Ethnicity was the strongest predictor of S-25(OH)D in immigrants compared with non-immigrants. Other predictors such as consumption of dairy products (milk, cheese, and yogurt) and traveling to a sunny/warm climate in the two months before blood sampling were also strongly associated with differences in S-25(OH)D. Overall, the model showed a robust association between immigration status and concentrations of S-25(OH)D.

**Table 5.** Multivariate analysis-backward elimination method based on S-25(OH)D (nmol/L) and immigration status using Cycles 3 and 4 of Canadian Health Measures Survey data.

Adjusted linear regression for age, sex, income, education, BMI, season, sun exposure, sunscreen use, country of birth, melanin levels, ethnicity, vitD-medication/supplements, and food consumption variables. Significant at *p* ≤ 0.05. BMI, body mass index; SE, standard error; CI, confidence interval.

#### **4. Discussion**

This is the first national Canadian study to report vitD status among immigrants from different ethnic groups and origins compared with non-immigrants and has a global impact. A previous systematic review noted the need to assess vitD status and its determinants (including lifestyle factors) among subgroups living in the same country [35]. Moreover, research has highlighted the importance of comparing the same generation of immigrants rather than an aggregated generation [22], and gathering evidence, and formulating recommendations specific to sub-populations that may differ from the overall immigrant population [16].

The higher overall prevalence of vitD insufficiency among immigrants than nonimmigrants in this study was consistent with global evidence suggesting that immigrants in Western countries have lower S-25(OH)D levels than non-immigrant populations [8,10,36,37]. Our multivariate analyses found ethnic background, having traveled to a sunny/warm climate, and low dairy product consumption were strong predictors of low S-25(OH)D levels among immigrants in Canada. Consistent with our findings, associations between S-25(OH)D levels and low income, winter season, low sunlight exposure, conservative dress, BMI (obesity), vitD supplements use, low physical activity, less traveling to a sunny/warm climate, non-white ethnicity, place of birth, and skin pigmentation were reported in previous studies [1,10,18].

While females are usually at higher risk for vitD deficiency [8,36–38], females in our study had markedly higher serum S-25(OH)D levels than males. In 2015, Statistics Canada reported that females had more elevated serum of vitD than males, more frequently using supplements than males (41% vs. 28%, respectively), and nearly 85% of vitD supplement users were above the cut-off (<50 nmol/L) compared with non-users (59%) [15]. McCormack et al. 2017, reported that females ≥ 19 years in Canada were using nutritional supplements (including vitD) more than males [39]. However, the higher levels of S-25(OH)D could be partly explained by the finding that females used vitD supplements more than males (66% vs. 34%, *p* = 0.019) (data not shown). In compliance with our finding, studies demonstrated that women who wear concealing clothes had lower S-25(OH)D levels than women dressed according to Western-style. It was well documented that the type of clothing determines the degree of sun exposure and supports/eliminates vitD's epidermal synthesis [8,10,18,40,41]. In this study, the effect of dress style on S-25(OH)D levels reduced (95% CI: −0.67 15.82; *p* = 0.070) in winter when both immigrants and nonimmigrants wear winter clothes that cover all the body, while the difference remained statistically significant for spring, summer, and fall which may be due to different type of clothing during these seasons.

The current study found that immigrants with low household income were more linked with lower S-25(OH)D levels. A global vitD review reported that higher family income in developing countries was found inversely associated with hypovitaminosis D [10]. Other studies confirmed that lower-income immigrants were found with lower levels of S-25(OH)D than higher incomes [42,43]. Consistent with our findings, global and Canadian vitD studies reported that obese adults had significantly lower S-25(OH)D than normal/underweight and overweight [1,8,15,44]. Studies suggested that this may be due to vitD displacement in adipose tissue, which results in lower circulating S-25(OH)D levels in the blood [8].

Dairy products contain essential nutrients including vitD and calcium. Previous studies suggested insufficient consumption of these products was strongly associated with lower S-25(OH)D levels [10,42,45,46]. Furthermore, milk in Canada is fortified with vitD, indicating a possibly magnified contribution of dairy products to serum vitD for both immigrant and non-immigrant consumers. Nonetheless, consumption of fortified milk and dairy products was relatively higher in non-immigrants than immigrants, which may partially explain the difference in S-25(OH)D levels.

Canada regions have different weather conditions; nonetheless, it is expected that days in fall and winter are shorter than in summer and spring, with fewer hours of sunlight overall in Canada. In the present study, immigrants had less sun exposure than nonimmigrants and had lower S-25(OH)D levels in winter than in summer. The seasonal variation in S-25(OH)D between immigrants and non-immigrants may be explained by the role of ultraviolet-B (UVB) light exposure in the endogenous synthesis of vitD. Previous studies also reported seasonal variations in vitD status [10,19,47]. Other studies reported the lowest levels of S-25(OH)D in winter [8,48], which was consistent with our findings.

The non-sunscreen users had a higher prevalence of deficiency and insufficiency in vitD status than did the frequent sunscreen users. However, such difference could be partly explained by the fact that sunscreens are used as photoprotector as it minimizes ultraviolet-B (UVB) light exposure, hence lowers endogenous vitD activation, and thus increases the likelihood of developing vitD deficiency [49]. Nonetheless, in practice, sometimes the irregular use of sunscreen or inadequate amounts may not be enough to secure the body from all UVB light. Furthermore, sunscreen users might feel sunburn protected by blocking UVB light and, hence, tend to expose themselves more to the sun than non-users, increasing S-25(OH)D levels. In line with our finding, 25% of studies in a recent review concluded that sunscreen use was associated with higher S-25(OH)D concentration, while 10% of studies reported lower levels and 35% with no association [49,50]).

Our data were obtained from immigrants from more than 150 countries, over 13 major ethnic groups, and therefore present a unique contribution to understanding Canada's ethnocultural diversity concerning vitD status. Irrespective of the length of time living in Canada, immigrants who were born in Africa and Asia had lower S-25(OH)D levels than those born in North America. The lowest levels by country of birth were among immigrants born in Algeria, Lebanon, and Morocco. In terms of ethnic origins, the lowest S-25(OH)D levels were in Japanese, followed by Arab and Southeast Asian ethnic groups. Our adjusted linear regression model indicated ethnicity was the most important indicator of low S-25(OH)D levels among immigrants compared with non-immigrants. Similarly, previous research reported vitD deficiency was more common in people from specific racial backgrounds. For example, Asian (South Asia, Southeast), African, and Middle Eastern immigrant populations had lower S-25(OH)D than their counterparts in Western countries [8,10,48]. Middle Eastern immigrants (including Lebanese and Iranian people) were more frequently reported to have deficient/insufficient levels than other ethnic groups and non-immigrant populations in Western countries [10]. A systematic review of 112 studies (168,389 participants from 44 countries) reported insufficient levels in one-third of the included studies, with the highest levels among participants living in North America and lower levels among those living in the Middle East and African regions [35].

Dark-skinned individuals have a rich amount of melanin, which acts as a biological shield against UV radiation [8]. In the present study, skin pigmentation was used to rank

participants based on melanin index values, where higher degrees of melanin indicated darker skin pigmentation. We found that immigrants had higher melanin levels than non-immigrants. Brook et al. reported the median 25(OH)D concentrations were higher in whites compared with non-whites in Canada [51] and several other studies have documented associations between skin pigmentation, ethnicity, geographical origins, and S-25(OH)D levels [8,18,52]. Research suggests that dark-skinned immigrants are at higher risk for vitD deficiency the longer time they spend in the host country [18]. In contrast, we found immigrants had markedly higher S-25(OH)D levels the longer they had lived in Canada after immigration. However, integration into a new life in Canada may reflect physical and behavioral adaptation to the surrounding environment, including lifestyle, diet, and climate changes. Immigration studies on acculturation and vitD found the length of time since immigration was a crucial indicator of lifestyle acculturation, and higher acculturation levels were associated with significantly higher S-25(OH)D levels [21,53]. However, we found immigrants had less sun exposure and less frequent consumption of vitD-rich foods than non-immigrants, and the length of time since immigration reflected reasonable lifestyle acculturation in Canada. Moreover, genomic studies showed that vitD interacts and influences the human genome through vitD receptor-mediated gene regulation and drives evolution for genomic adaptations in the context of skin color lightening [54]. The biological adaptation process is part of micronutrient gene-regulation that supports vitD3 synthesis in regions with lower UVB radiation [54]. Therefore, epigenetic adaptations may partially explain why immigrants in Canada were prone to vitD deficiency in the short term after immigration, and levels of S-25(OH)D increased over time. Furthermore, our study showed that ethnic differences played a fundamental role in S-25(OH)D levels. Common genetic polymorphisms in the vitD receptor and vitD-binding protein may partly explain differences in vitD status between different ethnicities [55]. Therefore, vitD insufficiency and deficiency among immigrants may be associated with genetic variations combined with dietary and other environmental factors; this issue warrants further investigation.

To our knowledge, this was the first study assessing S-25(OH)D levels among Canadian immigrants' different ethnic groups and origins compared with the non-immigrant population at the national level. The findings highlighted the need for further research investigating immigrants' health deterioration in the context of vitD. In addition, building knowledge of the relationship between health deterioration and vitD status along with similar biomarkers has important implications for responding to chronic and infectious diseases, including COVID-19.

Our study should be interpreted with consideration of its strengths and limitations. The CHMS data have many strengths. First, the CHMS provided the first national, comprehensive, and representative data for the Canadian population on vitD. Moreover, it used several clinical and laboratory measures to overcome the limitations of self-reported data [27]. Second, the study design and large sample size provided a nationally representative sample of immigrants in Canada. Third, it captured the ethnocultural diversity of immigrants from more than 150 countries and represented more than 13 major ethnic groups. Fourth, reported melanin level is a reliable indication of skin pigmentation. Fifth, S-25(OH)D levels were collected over the entire year, which enabled adjustment for seasonal variations in UVB light exposure, and offered a more accurate and representative measure of S-25(OH)D at the national level. Finally, the data were validated against other national data such as the Canadian Community Health Survey, Census data, and the 2011 National Household Survey [27]. However, immigrants were identified as landed people without detailed information about the immigration-specific status (e.g., refugees or asylum seekers) [27]. Research suggests refugees have a higher risk for vitD deficiency than other population groups [18,19,56,57]. The lack of data on refugees and asylum seekers limited further subgroup analyses. Another limitation of CHMS data is that the levels of biologically activated form 1,25(OH)2Dwas not measured in the study participants.

#### **5. Conclusions**

In conclusion, vitD deficiency is more common among Canadian immigrants than non-immigrants. Immigrants originating from African (Algeria and Morocco) and Asian (Lebanon) countries have a particularly low mean of S-25(OH)D. This study highlights ethnicity as a pivotal predictor of immigrants' lower S-25(OH)D levels; the Japanese, Arab, and Southeast Asian ethnic groups have a high prevalence of vitD deficiency. Short-term prevention strategies in community and clinical settings may be warranted, including vitD supplementation and awareness for people at higher risk. A long-term intervention plan may include developing/updating Canadian immigrant guidelines to incorporate evidence-based improvement strategies for vitD implementation.

**Author Contributions:** S.Y. and G.A.W. conceived the study. With close supervision from G.A.W., S.Y. managed the data, verified the analytical methods, and prepared the tables and figures. A.H. verified the underlying data at the Research Data Center. S.Y. and M.F. interpreted the results and prepared the final draft. All authors including D.M., I.C. and M.P. contributed to the design and analysis of the research, and the substantive review of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Institutional Review Board Statement:** All components of the national survey were reviewed and authorized annually by the Health Canada/Public Health Agency of Canada Research Ethics Board (REB# 2005-0025). The CHMS was approved by the Health Canada Research Ethics Board.

**Informed Consent Statement:** Written consents were collected by Statistics Canada from all participants for a personal interview, physical measures, and biospecimens collection.

**Data Availability Statement:** Data described in the manuscript, codebook, and analytic code will not be made publicly available because the data are confidential national survey data hosted by Statistics Canada.

**Acknowledgments:** The authors acknowledge the team from the Carleton, Ottawa, Outaouais Local Research Data Centre for assistance with access to CHMS data. CHMS data were obtained and supported by funding to the Canadian Research Data Centre Network from the Social Sciences and Humanities Research Council, the Canadian Institute for Health Research, the Canadian Foundation for Innovation, and Statistics Canada. Although the research and analysis were based on data from Statistics Canada, the opinions expressed do not represent the views of Statistics Canada.

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

#### **Appendix A**

Appendix A: Supporting information regarding the steps used to combine Cycles 3 and 4, and detailed information about the calculation of the variables of interest (Table A1).

**Table A1.** Variables of interest in Cycles 3 and 4 of the Canadian Health Measures Survey.

**Table A1.** *Cont.*

**Table A1.** *Cont.*

#### *Appendix A.1. Combining Cycles 3 and 4*

The CHMS is the first national population-based survey on vitamin D (vitD) conducted in Canada. Data were collected in six cycles biannually between 2007–2019. Compared with other cycles, Cycle 3 (2012–2013) and Cycle 4 (2014–2015) are considered the most thematically consistent in terms of vitD-related sociodemographic characteristics, lifestyle factors, vitD-rich foods consumption, and behavioral, environmental, and biological determinants. Immigrant status was defined as being born outside Canada [27]. In this study, we combined data from Cycles 3 and 4 to increase the sample size and provide a more stable and precise estimate of sampling variability at the national level than was possible using a single data cycle estimate. The combination of the two cycles followed Statistics Canada's guideline for combining CHMS cycles. As recommended, we used Bootstrap 1–Bootstrap 500, full-weight or applicable weight, and the required degrees of freedom to correct variance estimations. Therefore, the pre-specified combined full-sample weights and degrees of freedom for the two cycles were used in our analyses. The single-cycle weight was dropped from the dataset as it was a cycle-specific weight. The combined response rate (response rate for all study components, such as household visit, blood samples, and activity monitor) at the Canadian level for Cycle 3 was 51.7%, and that for Cycle 4 was 53.7% [33,34].

#### *Appendix A.2. Population, Comparators, Measures, and Outcome*

The population of interest in this study was first-generation Canadian immigrants (foreign-born) from different origins and ethnicities. The comparators were non-immigrants (native-born population) and the white ethnic grouping. The S-25(OH)D was measured using chemiluminescence immunoassay technology (DiaSorin®, Ltd., Stillwater, Minnesota, USA). The analytical detection limit for S-25(OH)D was 10–375 nmol/L. The inter-assay coefficients of variation for the S-25(OH)D was 13.0% and the precision for <20 nmol/L, 20–100 nmol/L and >100 nmol/L levels were 15.0%, 10.0% and 12.0%, respectively [45].

The results are presented as weighted means or proportions only. The weighted results and proportions were based on the total number of 11,579 participants. The number of participants for each group cannot be published because of Statistics Canada's restrictions policy relates to publishing weighted results. The weighting technique for complex survey results refers to statistical adjustments that have been used to correct and improve the accuracy of the survey estimates. The survey weights adjust for unequal probabilities of selection that often have occurred during sampling design and to help compensate for survey non-response weights. Thus, by using the survey weights to create estimates should yield approximately unbiased national prevalence estimates and reduce non-response bias that can be introduced due to random missing values. The study outcomes were the serum 25(OH)D levels and vitD status.

#### *Appendix A.3. Variables of Interest*

The data manipulation and all derived variables were consistent with Statistics Canada's instructions based on the specific cycle user guide, data codebook, instructions for combining more than one cycle, and the derived variable specifications [33]. The CHMS microdata are hosted in secure centers overall in Canada. These data were accessed and analyzed at the Carleton, Ottawa, Outaouais Local Research Data Centre (COOLRDC), located in the Morisset Library at the University of Ottawa.

#### **Appendix B**

Appendix B: supporting information including (Tables A2–A6).

This Appendix sets out the steps followed in combining Cycles 3 and 4 of the Canadian Health Measures Survey (CHMS), variables of interest, critical data manipulation stages, and other methodological aspects.

This Appendix contains supplementary material for the manuscript entitled "*Vitamin D Status among First-Generation Immigrants from Different Ethnic Groups and Origins: An Observational Study using the Canadian Health Measures Survey.*"

**Table A2.** Weighted prevalence and mean S-25(OH)D (nmol/L) levels and basic study characteristics using Cycles 3 and 4 of Canadian Health Measures Survey data.

SE, standard error.

*Nutrients* **2021**, *13*, 2702

† Reference value; SE, standard error; CI, confidence interval; BMI, body mass index. *p*-values: \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001.

**Table A5.** Weighted means for S-25(OH)D (nmol/L) by season and month of blood test using Cycles 3 and 4 of Canadian Health Measures Survey data.

† Reference value; SE, standard error; CI, confidence interval; *p*-values ≤ 0.05.

**Table A6.** Weighted mean S-25(OH)D concentrations (nmol/L) by immigration status and season using Cycles 3 and 4 of Canadian Health Measures Survey data.

**Figure A2.** Weighted prevalence of S-25(OH)D status for immigrants by region of birth using Cycles 3 and 4 of Canadian Health Measures Survey data.

**Figure A3.** Weighted prevalence of S-25(OH)D status by country of birth using Cycles 3 and 4 of Canadian Health Measures Survey data.

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## *Article* **Vitamin D, Vitamin D-Binding Proteins, and VDR Polymorphisms in Individuals with Hyperglycaemia**

**Rajiv Erasmus 1, Setjie Maepa 1, Ian Machingura 2, Saarah Davids 2, Shanel Raghubeer 2,\* and Tandi Matsha 2**

**Abstract:** Vitamin D reportedly plays an important role in the pathogenesis of diabetes mellitus; however, this role is unclear and debated. This study investigated the association between 25(OH) vitamin D, vitamin D-binding proteins, and vitamin D receptor (VDR) polymorphisms in healthy individuals and those with prediabetes and type 2 diabetes mellitus (T2D) from South Africa. A crosssectional study was conducted involving subjects of mixed ancestry aged ≥20 years. Males presented with higher mean 25(OH) vitamin D levels than females, while females exhibited significantly higher serum vitamin D-binding protein levels. Significant differences in mean 25(OH) vitamin D levels were observed in normo-glycaemic, prediabetes, screen-detected DM, and known DM individuals. Vitamin D receptor SNPs *Fok1* and *Taq1* were not associated with glycaemic status. *Fok1* was not associated with 25(OH) vitamin D deficiency, while *Taq1* was associated with vitamin D insufficiency. This study showed a high prevalence of vitamin D deficiency/insufficiency in this South African population, with decreased vitamin D levels observed in hyperglycaemic individuals, which was not linked to either vitamin D-binding protein or polymorphisms in *Fok1* of the *VDR* gene. These results may be used as a platform for further research into diagnosis and treatment of hyperglycaemia.

**Keywords:** hyperglycaemia; South Africa; vitamin D; vitamin D receptor; vitamin D-binding protein

#### **1. Introduction**

The role of vitamin D in non-communicable chronic diseases and the potential impact of vitamin D supplementation as a preventive and therapeutic measure is controversial and debated [1–4]. In cross-sectional epidemiological studies, insufficient vitamin D status is associated with the development of obesity, metabolic syndrome, and type 2 diabetes (T2D) in several but not all reports [5–8]. A recent systematic review reported that vitamin D supplementation at a minimum dose of 100 μg/d (4000 IU/d) may significantly reduce serum fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c), and homeostatic model assessment of insulin resistance (HOMA-IR) index in type 2 diabetic patients [9]. However, in a randomized trial, Pittas et al. concluded that vitamin D supplementation does not lower the risk of T2D [8]. Recently, Pilz et al. critically appraised several vitamin D randomized controlled trials and suggested that many researchers should carefully investigate the cohorts included in these studies, as cohort choice may bias the results obtained during these studies [10]. Furthermore, Xu et al. reported on a greatly improved sample size and concluded that genetically increased vitamin D concentration decreased T2D risk, suggesting that vitamin D supplementation deserves further investigation in interventional studies [11].

Vitamin D-binding protein is a serum glycoprotein, which is the major carrier protein of vitamin D sterols, and is essential for the intracellular metabolism of vitamin D.

**Citation:** Erasmus, R.; Maepa, S.; Machingura, I.; Davids, S.; Raghubeer, S.; Matsha, T. Vitamin D, Vitamin D-Binding Proteins, and VDR Polymorphisms in Individuals with Hyperglycaemia. *Nutrients* **2022**, *14*, 3147. https://doi.org/10.3390/ nu14153147

Academic Editor: Adrian Gombart

Received: 29 June 2022 Accepted: 28 July 2022 Published: 30 July 2022

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**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

Variations in vitamin D-binding proteins are postulated to influence the amount and activity of vitamin D, which in turn affect insulin secretion, β-cell dysfunction, and glucose metabolism [12]. Vitamin D exerts its effects on target tissues by binding to the cytosolic/nuclear vitamin D receptor (VDR), a member of the steroid/thyroid hormone receptor family. The VDR is expressed in the pancreas, and four polymorphisms of the vitamin D receptor, namely *FokI*, *BsmI*, *ApaI*, and *TaqI*, have been identified to be associated with insulin secretion and sensitivity. However, some studies have found no associations with these polymorphisms [13].

The mixed-ancestry population of Bellville South, Cape Town, exhibits a high prevalence of T2D [14], and vitamin D deficiency may contribute to the high prevalence of diabetes observed in this population group. In this study, we investigated the association between 25(OH) vitamin D levels, vitamin D-binding protein, and VDR polymorphisms in subjects with prediabetes and T2D in a mixed-ancestry South African population, as research is lacking in this population group.

#### **2. Materials and Methods**

#### *2.1. Ethical Approval*

The study forms part of the ongoing Vascular and Metabolic Health study (VMH), which received ethical approval from the research ethics committees of the Cape Peninsula University of Technology (CPUT) and Stellenbosch University (NHREC: REC-230 408–014 and N14/01/003, respectively). The current study also received ethical approval from the Stellenbosch University Health Research Ethics committee (0719) and Cape Peninsula University of Technology, Faculty of Health and Wellness Sciences Research Ethics committee (CPUT/HW REC2015/H01). Written informed consent was sought from all study participants following explanation of study procedures in their language of choice. All methods were performed in accordance with the Declaration of Helsinki and all relevant regulations.

#### *2.2. Study Population and Design*

This cross-sectional study comprised 1989 participants of mixed ancestry aged ≥ 20 years residing in Bellville South, Cape Town, South Africa. For the final analysis, individuals with incomplete data, acutely ill individuals, and pregnant females were excluded from the study. Therefore, the final sample size for this study was 968, from a total of 1989 participants. A detailed description of the survey and procedures conducted in this study have been published [14].

#### *2.3. Clinical Data*

The demographic and clinical data have been previously described, and were collected using a questionnaire [15]. The weight was measured to the nearest 0.1 kg using the Omron body fat meter HBF511 digital bathroom scale with participants wearing light clothing without shoes. The stadiometer was used to measure body height to the nearest centimetre with the study participants standing on a flat surface. The body mass index was calculated as weight per square meter (kg/m2). The waist circumference was measured using a nonelastic tape at the level of the narrowest part of the torso, as seen from the anterior view, whilst in obese participants the narrowest circumference between the ribs and the iliac crest were measured. Hip circumference was measured at the maximal circumference over the buttock using a non-elastic tape. Body mass index was used to classify participants as underweight (<18.5 kg/m2), normal (18.5–24.99 kg/m2), overweight (≥25 kg/m2), or obese (≥30 kg/m2) according to the World Health Organisation (WHO) criteria [16]. Participants who did not have a medical history of diagnosis with diabetes mellitus underwent a 2 h oral glucose tolerance test (OGTT), as recommended by the WHO. The OGTT was used to group the study participants as normoglycaemic or prediabetic (including impaired fasting glycaemia, impaired glucose tolerance, or a combination of both) using the WHO criteria [17]. The Joint Interim Statement of the International Diabetes Federation Task Force on Epidemiology and Prevention, National Heart, Lung and Blood Institute, American

Heart Association, World Heart Federation, International Atherosclerosis Society, and International Association for the Study of Obesity (JIS) was used to classify metabolic syndrome (MetS) [18].

#### *2.4. Biochemical Analysis*

Blood samples were collected from all participants after fasting overnight. Plasma glucose was measured using the hexokinase method (Cobas 6000, Roche Diagnostics; Mannheim, Germany), HbA1c using high-performance liquid chromatography (HPLC; Bio-Rad Variant Turbo, Bio-Rad, South Africa), which was National Glycohaemoglobin Standardization Program (NGSP) certified, insulin using the paramagnetic particle chemiluminescence assay (Beckman DXI, Beckman Coulter, South Africa), fructosamine using a colorimetric test with nitro blue tetrazolium (Cobas c311, Roche Diagnostics), lowdensity lipoprotein cholesterol (LDL-C; mmol/L) using enzymatic selective protection-end point (Beckman AU, Beckman Coulter), HDL-C (mmol/L) using enzymatic immuneinhibition—end point (Beckman AU), and triglycerides (TG; mmol/L) using glycerol phosphate oxidase-peroxidase—end point (Beckman AU). The 25(OH) vitamin D levels were measured using the paramagnetic particle chemiluminescence test (Beckman DXI), and vitamin D-binding protein (VDBP) was determined using the Human Vitamin D BP Quantikine ELISA kit (DVDBP0; R&D Systems, Minneapolis, MN, USA).

#### *2.5. Definition of Vitamin D Deficiency*

Vitamin D deficiency was defined using either the 2011 Endocrine Clinical Society Practice Guidelines as 25 hydroxyvitamin D (25(OH) vitamin D) below 20 ng/mL (50 nmol/L) and vitamin D insufficiency as 25(OH) vitamin D between 20 and 29 ng/mL (50–75 nmol/L) [19] or the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets, with vitamin D deficiency defined as 25(OH) vitamin D below 12 ng/mL (30 nmol/L) and vitamin D insufficiency as 25(OH) vitamin D between 12 and 20 ng/mL (30–50 nmol/L) [20].

#### *2.6. Genetic Analysis*

Genomic DNA was extracted from whole blood samples collected in EDTA tubes using the salt extraction method, then quantified using the NanoDrop ND-1000 instrument (Nanodrop Technologies, Wilmington, USA). *VDR* single-nucleotide polymorphisms, *Fok1* (*rs2228570*), *Apa1* (*rs7975232*), and *Taq1* (*rs731236*), were genotyped using high-throughput real-time polymerase chain reaction on the Bio-Rad Optica platform (Bio-Rad, Hercules, CA, USA) using TaqMan™ genotyping assays. Primers were predesigned TaqMan™ SNP genotyping assays. All primers and kits comply with the minimum information for publication of quantitative RT-PCR experiments (MIQE). All primer sequences can be accessed via the Thermo Fisher Scientific (Waltham, MA, USA) website. Thereafter, all samples were submitted to Inqaba Biotechnical Industries (Pretoria, South Africa) for further verification by an independent laboratory. The conventional polymerase chain reaction followed by direct DNA sequencing was performed for analytical validation of genotyping.

#### *2.7. Data Analysis*

Data were analysed using Statistica 13.3 (StatSoft, Pretoria, South Africa). Categorical variables were summarized as count and percentages, while quantitative variables were indicated as mean (standard deviation) or median (25th–75th percentiles). Variable comparisons across the glycaemic status were conducted using the chi-squared test. The Pearson chi-square test was used to determine association between single-nucleotide polymorphism genotypes and/or allele frequencies and vitamin D deficiency, obesity, and glycaemia categories. A multiple linear regression model was used to establish possible associations between vitamin D and other test results. A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Participant Characteristics and Vitamin D Deficiency*

A total of 968 study participants were recruited, 79.2% of which were female. Table 1 indicates the characteristics of study participants and vitamin D categories (deficiency, insufficiency, and optimal) using the 2011 Endocrine Clinical Society Practice Guidelines (*p* = 0.004) and the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets (*p* = 0.003), varied according to sex. Male participants were older, exhibited greater waist/hip ratios and higher glucose/insulin ratios and levels of serum creatinine, urine creatinine, aspartate aminotransferase, serum albumin, urine sodium, and cotinine. Female participants exhibited greater waist and hip circumference measurements, higher levels of two-hour postprandial blood glucose, HbA1c, fasting blood insulin, two-hour postprandial blood insulin, HDL cholesterol, LDL cholesterol, cholesterol, parathormone, calcium corrected, and phosphate, and included a greater percentage of smokers. Vitamin D and vitamin D-binding protein levels varied according to sex, with males exhibiting higher mean 25(OH) vitamin D levels than females (24 ± 8 vs. 22 ± 8 ng/mL, *p* = 0.0006, respectively), whilst females displayed significantly higher serum vitamin D-binding protein levels (323 ± 81 vs. 306 ± 74 μg/mL, *p* = 0.007).

**Table 1.** Participant characteristics categorized according to sex.

**Table 1.** *Cont.*

DM, diabetes mellitus; BMI, body mass index; WaistC, waist circumference; HipC, hip circumference; WHR, waist-to-hip-ratio; Vitamin D BP, vitamin D binding protein; FBG, fasting blood glucose; BG, blood glucose; HbA1c, glycated haemoglobin; FBI, fasting blood insulin; BI, blood insulin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Chol, cholesterol; GT-S glutamyl transferase-serum; ALT, alanine aminotransferase; SGPT, serum glutamic-pyruvic transaminase; AST, aspartate aminotransferase; SGOT, serum glutamic-oxaloacetic transaminase; MDRD, modification of diet in renal disease; CRP, c-reactive protein. Bold *p* values indicates significant differences in the analysed data.

Using the 2011 Endocrine Clinical Society Practice Guidelines, the prevalence of vitamin D deficiency and insufficiency amongst all participants was 44.5% and 42.5%, respectively. As expected, when using the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets, the prevalence of vitamin D deficiency and insufficiency amongst all study participants was 5.6% and 39.2%, respectively (Table 2). The Institute of Medicine (IOM) recommended a minimum vitamin D level of ≥20 ng/mL for adults in 2011; thus, the mean vitamin D level amongst all study participants fell within this recommended level [1,21].

**Table 2.** Vitamin D deficiency, insufficiency, and optimal level cut-offs used to assess vitamin D status.

#### *3.2. Glycaemic Status and Vitamin D Deficiency*

Table 3 shows the characteristics of the study participants according to their glycaemic status. Using the 2011 Endocrine Clinical Society Practice Guidelines for vitamin D deficiency, vitamin D varied according to the glycaemic status, with normoglycaemic participants displaying higher vitamin D levels than prediabetes mellitus, screen-detected diabetes mellitus, and known diabetes mellitus participants (*p* = 0.002).

Using the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets, there was no significant difference in the prevalence of deficient, insufficient, and optimal 25(OH) vitamin D. Further, levels of vitamin D-binding protein were significantly decreased in the deficient vitamin D group compared to the insufficient vitamin D group and the optimal vitamin D group (*p* = 0.007), according to the 2011 Endocrine Clinical Society Practice Guidelines for vitamin D deficiency (Figure 1). Similarly, vitamin D-binding protein levels were significantly decreased in the deficient vitamin D group compared to insufficient and sufficient vitamin D groups (*p* = 0.003), according to the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets vitamin D deficiency (Figure 2).

#### *3.3. Genotype Distribution of VDR Polymorphisms*

The Hardy–Weinberg Equilibrium (HWE) test was used to determine SNP frequency. Table 4 shows genotype distribution and minor allele frequencies within the study population. The allele percentage was calculated at 26.5% for *Fok1* (*rs2228570*; HWE *p* = 0.1), 28.9% for *Taq1* (*rs731236*; HWE *p* = 0.6, and 39% for *Apa1* (*rs7975232*; HWE *p* = 0.02). In the dominant (GG versus AA + AG), recessive (GG + AG versus AA), and additive models for SNP *rs2228570*, the determinants, sex, age, glycaemic status, MetS, and vitamin D, were not significant (Table 5). However, results from the additive model indicate that obesity was a significant determinant, with a two-fold increase in the GG genotype [OR (95% CI): 2.04 (1.02; 4.10), *p* = 0.045].

Table 6 shows that in the dominant (AA versus GG + AG), recessive (AA + AG versus GG), and additive (AA vs. AG vs. GG) models for the SNP *rs731236*, the determinants, sex, age, glycaemic status, and MetS, were not significant. However, in the recessive model, the SNP *rs731236* was associated with insufficient vitamin D (*p* = 0.04). In the additive model, there was a lower likelihood of patients being overweight [OR (95% CI):0.65 (0.43; 0.97), *p* = 0.04] in the genotype AG, while the genotype AA had a 1.82 likelihood of being vitamin D-insufficient [OR (95% CI):1.82 (1.07; 3.07), *p* = 0.03].

**Table 3.** Participant characteristics according to glycaemic status.

**Table 3.** *Cont.*

**Figure 1.** Using the Endocrine Clinical Society Practice Guidelines for vitamin D deficiency, vitamin D-binding protein (μg/mL) levels were found to be significantly decreased in the deficient vitamin D group, mean ± SD, 311 ± 75 (μg/mL) (*N* = 431), compared to the insufficient vitamin D group, 327 ± 85 (*N* = 408), and the optimal vitamin D group, 328 ± 75 (*N* = 126), (*p* = 0.007). BP, binding protein.

**Figure 2.** Using the Global Consensus Recommendations on Prevention and Management of Nutritional Rickets vitamin D deficiency, vitamin D-binding protein (μg/mL) levels were found to be significantly decreased in the deficient vitamin D group, mean ± SD, 300 ± 57 (μg/mL) (*N* = 54), compared to the insufficient vitamin D group, 312 ± 77 (*N* = 379), and the sufficient vitamin D group, 327 ± 83 (*N* = 126), (*p* = 0.003). BP, binding protein.

**Table 4.** Genotype distributions and minor allele frequencies (HWE: Hardy–Weinberg Equilibrium).

Bold *p* values indicates significant differences in the analysed data.

*Nutrients* **2022** , *14*, 3147

*rs731236*.

*Nutrients* **2022**

, *14*, 3147

#### **4. Discussion**

Vitamin D deficiency is one of the most prevalent nutritional deficiencies in the world. Vitamin D, which was previously known to be involved only in calcium homeostasis, is now known to have several other functions in the human body [22]. Subclinical and asymptomatic vitamin D deficiency is associated with increased risk of multiple malignancies, metabolic and cardiovascular diseases, diabetes, and immune disorders [23]. Studies regarding vitamin D supplementation in African populations are limited [24,25]. Over the last decade, low vitamin D levels have emerged as a risk factor for T2D, but this has not been investigated in South African populations. In this community-based study, we examined the association between serum 25-hydroxy vitamin D levels and glycaemic indicators in diabetic, prediabetic, and healthy subjects from a population at high risk of developing diabetes residing in an urban area of Cape Town, South Africa [14]. Due to differences in opinions regarding cut-off levels of vitamin D deficiency [26], we used both the 2011 Endocrine Clinical Society Practice Guidelines and the more recent Global Consensus Recommendations on Prevention and Management of Nutritional Rickets to define vitamin D deficiency as either a 25(OH) vitamin D level below 20 ng/mL (50 nmol/L) or 12 ng/mL (30 nmol/L) [20]. Similar to reports from Germany and Japan, we found a mean 25(OH) vitamin D level of 22 ng/mL, which was within the optimal levels of the Global Consensus Recommendations, but considered to be insufficient according to the Endocrine Society Guidelines [19]. We observed significant differences in the overall prevalence of vitamin D deficiency, with 44.5% of the participants classified as deficient according to the Endocrine Clinical Society Practice Guidelines, but only 5.6% were found to be deficient when using the Global Consensus Recommendations. Only 13% had optimal levels, whilst 44.7% had sub-optimal levels when using the latter criteria.

We observed vitamin D deficiency in subjects with either prediabetes, screen-detected diabetes mellitus, or known diabetes mellitus compared to normoglycaemic subjects using the former criteria. Surprisingly, when using the Global Consensus Criteria, which uses a much lower cut-off to define vitamin D deficiency, the percentage of deficient subjects was similar in each of the glycaemic groups (Table 3). This raises an important question: what levels must we use to define vitamin D deficiency? When comparing our results with earlier studies that have used higher cut-offs endorsed by the Endocrine Society, similar conclusions were arrived at: vitamin D deficiency is associated with prediabetes and diabetes. In a study from Japan, which used a cut-off of 50 nmol/L, vitamin D deficiency was found in 54% of participants, whilst it was 90.9% when a cut-off of 75 nmol/L was used. The high prevalence of vitamin D insufficiency in Japanese populations was attributed to darker skin and rare use of vitamin D supplements [27]. Similarly, studies from Egypt and Bangladesh have reported lower vitamin D levels in T2D patients [28,29] compared to healthy controls. Further research is required regarding the influence of skin colour on vitamin D levels.

A study from China examined if higher plasma 25(OH) vitamin D concentrations were associated with lower risks of diabetes in 82500 participants and further tested the relevance of 25(OH) vitamin D in T2D subjects using genetically instrumented differences in plasma 25(OH) vitamin D concentrations to ascertain causality. The concordant results of both the observational and genetic studies indicate that a higher vitamin D status is associated with a lower risk of diabetes and provide support for a causally protective effect of higher vitamin D in the prevention of T2D [30]. Abbasi et al. showed that subjects with prediabetes and low circulating 25(OH) vitamin D levels were mostly insulin-resistant, had impaired β-cell function, and were most likely to develop T2D [31].

In our study, both obesity and overweight were commonly observed. A higher body mass index (BMI) has been associated with lower vitamin D levels. Obesity affects insulin secretion, tissue sensitivity to insulin, and systemic inflammation, but this may not account for differences seen in the levels of vitamin D deficiency between the glycaemic groups, as BMIs were similar. A meta-analysis that examined 55 observational studies showed an inverse relationship between vitamin D levels and BMI in both diabetic and non-diabetic subjects [32]. Studies in low- and middle-income countries have consistently demonstrated that women have lower average 25(OH) vitamin D levels than their male counterparts, which is largely thought to be due to differences in occupation, clothing, and cultural practices, which predisposes women in these countries to lower vitamin D status and is not related to biological differences in vitamin D metabolism between males and females [33]. Similarly, in this study we found lower vitamin D levels in females than in male study participants, although females displayed higher vitamin D-binding protein levels. These sex differences may partially be attributed to the higher BMIs observed in females. Surprisingly, no sex differences were observed in CRP levels. Several studies have suggested that the lower the vitamin D level, the greater the benefit of supplementation in preventing diabetes [34,35]. Thus, it may be prudent to consider the benefit of vitamin D supplementation in this population group, which is at high risk of developing diabetes.

The *VDR* gene is highly polymorphic, widely distributed, located on chromosome 12q13.1 [36], and controls genes related to bone metabolism, inflammation, oxidative damage, and chronic diseases [37]. Vitamin D and its receptor complex play a role in the regulation of insulin secretion from beta cells [38,39]. *VDR* gene variations are associated with the development, progression, and complications of T2D [40,41]. If the vitamin Dbinding protein gene is mutated, vitamin D would decrease in serum and target tissues, although sufficient sun exposure or supplementation may ameliorate this. Four common single-nucleotide polymorphisms of the *VDR* gene have been postulated to be associated with T2D in different ethnic populations, namely *FokI* (*rs2228570*), *BsmI* (*rs1544410*), *ApaI* (*rs7975232*), and *TaqI* (*rs731236*). The full-length human *VDR* gene is ~63.5 kb.

In this study, the *VDR* single-nucleotide polymorphisms *Fok1* (*rs2228570*) and *Taq1* (*rs731236*) were not associated with glycaemic status. *Fok1* was also not associated with vitamin D level, although *Taq1* was associated with insufficient vitamin D. Amongst a population in Saudi Arabia, no significant association between the *Fok1* and *Taq1* singlenucleotide polymorphisms and vitamin D deficiency was observed [42]. A study conducted in Russia showed no difference in serum 25(OH) vitamin D concentration between *Taq1* and *Fok1* genotypes [43]. However, in a population from Bangladesh at high risk of T2D, the *ApaI* polymorphism was associated with insulin secretion and a higher prevalence of vitamin D deficiency. The *ApaI* polymorphism was correlated with fasting blood glucose levels, and glucose intolerance was evident among individuals with symptoms of diabetes at the pre-diagnosis stage [44]. As ethnicity reportedly influences *VDR* gene variations [45], the variations observed in our study may be explained by this and the participants' exposure to environmental factors [46]. Our results indicate that *Fok1* was associated with obesity, similar to observations in T2D Egyptian patients. Patients with mutant recessive homozygous TT genotype C>T polymorphisms exhibited higher waist circumference and BMIs than individuals with the homozygous CC genotype [46]. In our study, the GG genotype of the *Fok1* polymorphism was associated with a two-fold increased risk of developing obesity, similar to subjects harbouring the T allele in Greece [47].

Our study, like others, is not without limitations. The comparison of vitamin D status between different studies is difficult due to the lack of an evidence-based consensus regarding optimal levels of serum 25(OH) vitamin D, since cut-offs used to evaluate vitamin D status vary across studies. Although serum 25(OH) vitamin D measurement is a valid and commonly used biomarker of vitamin D status, its measurement still lacks standardization. Thus, the measurement of 25(OH) vitamin D differs between studies due to differences in analytical methods, assays, and devices used [48]. There are also seasonal variations in serum 25(OH) vitamin D levels, with the highest levels observed towards the end of summer and lowest levels toward the end of winter, but tracking 25(OH) vitamin D concentration over time reveals that a single measurement of serum 25(OH) vitamin D at a given point provides an estimate of future 25(OH) vitamin D levels [10]. Furthermore, this study did not take dietary choices or physical activity into consideration. As such, behavioural differences may influence the interpretation of the results. Further studies are

required to determine the influence of diet in this cohort, as well as in a cohort with similar dietary preferences and levels of physical activity.

#### **5. Conclusions**

We observed that vitamin D deficiency or insufficiency is relatively common in this mixed-ancestry population from Cape Town. Furthermore, we found that vitamin D levels were decreased in individuals with hyperglycaemia, which was not linked to either vitamin D-binding protein or *Fok1* polymorphisms in the vitamin D receptor gene, although *Taq1* was associated with an insufficient vitamin D status. The mechanisms affecting lower vitamin D levels in individuals with hyperglycaemia require further investigation. Finally, a clinical trial of vitamin D supplementation to either revert prediabetes to normoglycaemia or prevent its progression to diabetes in this population group is highly desirable and recommended.

**Author Contributions:** Conceptualization, R.E. and T.M.; Data curation, S.D. and T.M.; Formal analysis, R.E., S.M., I.M. and S.D.; Funding acquisition, R.E. and T.M.; Investigation, R.E., S.M., I.M., S.D., S.R. and T.M.; Methodology, R.E., S.M., I.M., S.D., S.R. and T.M.; Project administration, S.R. and T.M.; Supervision, R.E. and T.M.; Visualization, S.D.; Writing—original draft, R.E., I.M. and S.R.; Writing—review and editing, R.E., S.M. and S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project was funded by the South African Medical Research Council with funds from the National Treasury under its Economic Competitiveness and Support Package (MRC-RFA-UFSP-01–2013/VMH Study) and the South African National Research Foundation (Grant no. 115450). Any opinion, finding, and conclusion or recommendation expressed in this material is that of the authors, and the Medical Research Council does not accept any liability in this regard.

**Institutional Review Board Statement:** The study forms part of the ongoing Vascular and Metabolic Health study (VMH), which received ethical approval from the research ethics committees of the Cape Peninsula University of Technology (CPUT) and Stellenbosch University (NHREC: REC-230 408-014 and N14/01/003, respectively). The current study also received ethical approval from the Stellenbosch University Health Research Ethics committee (0719) and Cape Peninsula University of Technology, Faculty of Health and Wellness Sciences Research Ethics committee (CPUT/HW REC2015/H01). Written informed consent was sought from all study participants following explanation of study procedures in their language of choice. All methods were performed in accordance with the Declaration of Helsinki and all relevant regulations.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The sequence data used to support the findings of this study have been deposited in the SRA BioProject database (accession number: PRJNA723337).

**Acknowledgments:** The authors would like to thank the community of Bellville South.

**Conflicts of Interest:** The authors declare no conflict 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**

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**Abstract**

There has been increased concern on many levels focused on the environmental and occupational exposure of heavy metals and their impact on disease, specifically the carcinogenic potential inducing cancer in humans. Because the impact of heavy metals on human health continues to be a major health concern, research continues to improve our understanding of the carcinogenic potential of these substances. Of particular concern have been human exposure to aluminum, arsenic, beryllium, cadmium, lead, mercury, nickel, and radium and their carcinogenic potential whether contact is via environmental or occupational exposure. This updated review focuses on the carcinogenic mechanisms heavy metals use to induce malignant transformation of cells as well as addressing the overall environmental and occupational hazards of heavy metal exposure.

**Keywords:** heavy metals, carcinogenesis, human exposure, toxicity, mechanisms, remediation


*Model Development and Validation*

The landslide inventory data was randomly categorized into two datasets, i.e., training (70%) and testing (30%). The landslide susceptibility map based on 8 causative factors using AHP is depicted in Figure 10. The map was divided into 4 classes: very low (0–0.24), low (0.25–0.49), moderate (0.5–0.74) and high (0.75–1) according to natural breaks to define the class intervals in the susceptibility map. From total of 242 landslides, more than 78.1% falls under the high zone. Whereas 11.98% falls under the moderate zone and combined 9.92% falls under the low and very low zones (Table 5).

**Figure 10.** Landslide Susceptibility Map of the Study Region.

**Table 5.** Landslide Susceptibility Results for the Samdrup Jongkhar–Trashigang Highway Region.

Validation of the susceptibility maps was performed for randomly selected data from the inventory data using receiver operating characteristics (ROC). This is an effective way to analyze the quality of predictive techniques [81]. The ROC curve is plotted between true positive rate (sensitivity) on the Y

axis against false positive rate (specificity) on the X axis. The terms "sensitivity" and "specificity", which are used to plot ROC curves, are defined as follows.

$$\text{Sensitivity} = \frac{\text{TP}}{\text{TP} + \text{FN}} \tag{7}$$

$$\text{Specificity} = \frac{\text{TN}}{\text{FP} + \text{TN}} \tag{8}$$

where true positive (TP) is the number of actual landslides predicted correctly, and true negative (TN) is the total number of non-occurring landslides predicted correctly. False positive (FP) is the number of actual landslides inaccurately predicted as non-occurring landslides, and false negative (FN) is the number of non-occurring landslides inaccurately predicted as actual landslides. [82]. The area under the ROC curve (AUC) was also used to determine the quality of the prediction by analyzing the model's ability to forecast the occurrence or nonoccurrence of predefined events [83]. The results of the success rate curve of the AHP model had an AUC of 0.798, corresponding to a prediction accuracy of 79.8% (Figure 11).

**Figure 11.** Receiver operating characteristic (ROC) curve of the susceptibility map by AHP.

The results of pairwise comparison, priority estimation, and ranking of all the criteria could be applied to other study areas for susceptibility assessment. Several high-resolution satellite image datasets are required to better understand the locations and perform these assessments. In cases of unavailability of high-resolution satellite images, Google Earth images could be useful for preparation of some indicators. The use of Google Earth images could also be helpful for accurate identification of landslide locations and conduction of extensive field studies. All the requirements for more accurate analysis depend on study location tectonics, data availability, and proper methods. Therefore, based on local geology and tectonic conditions, these results could be transferable and applicable in other locations in both small- and large-scale areas.

### **6. Conclusions**

Landslides are the most frequently occurring natural hazards, especially in the Himalayan regions, which suffer from heavy monsoonal rainfall and subsequent landslides. In this study, the temporal probability of landslide events was determined using rainfall and landslide data from 2014–2017 along Samdrup Jongkhar–Trashigang highway in East Bhutan. The highway was divided into three zones based on land use, topography, and rain gauge coverage for the determination of temporal probability. Thereafter, a landslide susceptibility map was developed using AHP. The results of temporal probability

were validated with landslide event dataset of 2018 to understand the applicability of the model. The conclusions from the study can be summarized as follows.

The present study on rainfall threshold estimation and the development of a susceptibility map for eastern Bhutan along Samdrup Jongkhar–Trashigang highway is an important study in the context of the Bhutan Himalayas, for which study on both these aspects is lacking. The current study can be regarded as a preliminary step towards risk management, which could be supported by conducting a hazard and vulnerability assessment of the region. The temporal probabilities determined can be integrated with the susceptibility map to obtain a landslide hazard map. However, the results might be improved by increasing the number of landslide events and using precipitation data with a higher temporal resolution. The results from the present study also could prove helpful to civic authorities in identifying key sections of the road which are most vulnerable to landslides, and undertaking strict measures to prevent slope failures, strengthening the transportation network and saving human lives.

**Author Contributions:** Conceptualization, A.D. and R.S.; methodology, A.D. and R.J.; data curation, D.D.; writing—original draft preparation, A.D. and R.S.; writing—review and editing, B.P. and A.M.A.; supervision, B.P. and R.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Centre for Advanced Modelling and Geospatial Information Systems (CAMGIS) in the University of Technology Sydney (UTS) under Grants<PHONE_NUMBER>335 and<PHONE_NUMBER>357; Grant<PHONE_NUMBER>424, and Grant<PHONE_NUMBER>452. This research is also supported by Researchers Supporting Project number RSP-2019/14, King Saud University, Riyadh, Saudi Arabia. Also, the research was funded from BRACE project (NERC/GCRF NE/P016219/1).

**Acknowledgments:** The authors are thankful to National Center for Hydrology and Meteorology, Royal Government of Bhutan for providing rainfall data and the Border Roads Organization (Project DANTAK), Government of India for providing landslide data. Authors are also thankful to staff of College of Science and Technology, Royal University of Bhutan, who helped directly or indirectly while carrying out the present study. We also thank the editor and the reviewers for reviewing and suggesting valuable modifications, which has helped in improving the quality of this paper.

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


**2. The Effect of Government Response on the Infection Rate**

*2.1. Immediate Effects of Government Response on the Infection Rate*

Studies have shown that social interventions are needed to control the spread of epidemic diseases. Bauch and Galvani point out thatcontrol of the SARS coronavirus depended partly on the degree of acceptance of quarantine and isolation among the population; such acceptance is often determined by social norms [12]. In the book *The Rules of Contagion: Why Things Spread and Why They Stop*, Kucharski concluded that the factors that influence the reproduction number of an epidemic disease include duration, opportunities, transmission, and susceptibility [13]. In his view, curing a patient reduces the duration of infection, isolating a patient reduces the opportunities for infection, wearing a condom or mask reduces contagion, and vaccination reduces population susceptibility. Government response to COVID-19 consists of social interventions implemented to curb the spread of the disease. COVID-19 intervention policies are complex and vary between countries, but they can be broadly categorised into five major areas, namely, containment and closure, economic responses, health systems, vaccine policies, and miscellaneous policies [3]. Many studies have attempted to determine the most effective intervention policies. For example, Richard et al. examined the effects of four types of government response—event bans, school closures, bar and pub closures, and lockdown—and discovered that event bans and school closures directly reduced virus transmission, while the influence of a full lockdown was slightly delayed [14]. Scholars have used epidemiological data on COVID-19 and anonymised migration data to simulate outbreaks and intervention effects across China. A comparison of infections in Wuhan, Hubei province, with those in other cities in Hubei and cities in other provinces revealed that early detection and isolation were more effective than travel restrictions. Reducing social contact curbed the spread of COVID-19 and prevented or delayed the arrival of a second wave of the outbreak. The authors also found that although travel restrictions had not prevented the virus from spreading from Wuhan, they had prevented its wider geographical spread [4].

The degree of policy implementation is also an important predictor of the COVID-19 infection rate. An international comparative study using data from China, Italy, Brazil, Canada, the United Kingdom, and the United States found that the stringency of intervention policy implementation was negatively associated with the number of new cases. This study also found that the Chinese government had maintained strong prevention and control measures for the first 100 days of the outbreak, during which China had experienced a dramatic decrease in infections [3]. When the virus was first detected in Guangdong province, the province's health commission quickly activated a Level I emergency response and implemented a series of public interventions, including traffic restrictions, social distancing, home and centralised quarantines, medical resource mobilisation, and other prevention and control measures, which significantly restrained the local spread of the disease [15]. Differences in the degree of policy implementation may stem from differing individual responses to government policies or from differences in national policy environments, such as social norms, cultural traditions, the political atmosphere, and other macro-level factors that interact with government response [16,17]. In general, policies that are strictly enforced tend to bring about better results, especially in the early stages [3,4,6,14–16].

The timing of policy initiation is another important predictor of the effectiveness of government response to COVID-19. Take social distancing as an example. A series of studies found that isolating infected people decreased and delayed transmission as well as reducing the epidemic's peak [4,8,18–20]. Using counterfactual simulations, another study discovered that if the same restrictions on mobility had been implemented just one to two weeks earlier, a substantial number of cases and deaths would have been avoided. Specifically, 61.6% of the infections and 55% of the deaths reported nationwide by May 3, 2020 could have been avoided if these preventive and control measures had been implemented just one week earlier [4]. A study of the relationship between the first emergency quarantine policy in Portugal from 18 March to 2 May 2020 and the public's health behaviour showed that 79.8% of the participants, whose physical activity took place indoors, complied with the government quarantine measures and adapted their health behaviour [21]. Therefore, early social distancing plays a key role in relieving pressure on healthcare facilities and ensuring a sustained supply of healthcare resources. At this level, the timing of social distancing implementation is crucial to controlling large-scale outbreaks. Social distancing has been shown to reduce not only new cases but also cumulative cases. This implies that early government intervention may have some sustained effects, in that people became more aware of the virus during home isolation and were more likely to adopt protective action recommendations (PARs) after home isolation, thereby reducing their own infection rates.


**4. Discussion**

Matrix free [Ca2+] ([Ca2+]m) plays two important roles: (i) Activation of Ca2+-dependent dehydrogenases for oxidative phosphorylation at low concentrations [46]; and (ii) regulation of cytosolic Ca2+ by sequestration of excess Ca2+ at high concentrations [47]. Excessive accumulation of free [Ca2+]m is a leading factor in inducing mPTP opening. It is well established that repetitive mitochondrial Ca2+ loading triggers a gradual increase in [Ca2+]m, leading to a loss of IMM integrity that results in dissipation of ΔΨm and release of Ca2+. CsA is known to delay pore opening, in part, by inhibiting the PPIase activity of Cyp-D [31]. Whether CsA-mediated delay in mPTP opening involves regulation of [Ca2+]m by Pi-induced matrix Ca2+ buffering has not been addressed before. In this study, we investigated the effects of CsA on [Ca2+]m regulation during repeated Ca2+ loading and its functional significance in mPTP opening. Additionally, we determined if changes in [Ca2+]m induced by CsA correlated with changes in mitochondrial bioenergetics under identical experimental conditions and if matrix Pi was required for the observed CsA effects.

Since the key postulate was that CsA contributes to mitochondrial Ca2+ buffering, all experiments were performed in Na+-free condition to completely block NCLX as a route for efflux of excess matrix Ca2+. This allowed us to directly assess mitochondrial Ca2+ buffering capacity under different treatments. Our major findings during repetitive CaCl2 bolus challenges are: (i) CsA maintained basal ss[Ca2+]m owing to increased mitochondrial Ca2+ buffering capacity; (ii) the effectiveness of CsA to maintain basal ss[Ca2+]m correlates well with preserved mitochondrial bioenergetics; (iii) the buffering effect of CsA in a Pi-replete buffer was more pronounced than the known buffering effect of OMN+ADP; (iv) CsA-induced buffering was abolished in Pi-depleted mitochondria and Pi-free experimental medium. We conclude that the CsA-mediated delay in mPTP opening could, in large part, be attributed to CsA-induced activation of a Pi-dependent mitochondrial Ca2+ buffering system (MCBS), which maintains a low free [Ca2+]m and preserves mitochondrial bioenergetics.

#### *4.1. CsA-Mediated Inhibition of mPTP Opening Relates to the ss[Ca2*+*]m*

Using the two protocols (Figure 1A,B), we examined the changes in [Ca2+]e and [Ca2+]m in response to boluses of CaCl2 in the presence of vehicle (DMSO), CsA, ADP, OMN, or OMN+ADP over time. Our experimental approaches allowed us to define the contribution of CsA in the regulation of [Ca2+]m when CsA was given before the CaCl2 boluses (Protocol A) and at the threshold for pore opening under condition of increased free [Ca2+]m accumulation (Protocol B). Our results clearly indicate that the effect of CsA on delaying mPTP opening is due largely to its efficacy in maintaining free ss[Ca2+]m by activating the MCBS in a Pi-dependent manner, and thereby preclude early mitochondrial Ca2+ overload and delay induction of mPTP opening. Sustained low ss[Ca2+]e in the CsA-treated group indicated increasing mitochondrial Ca2+ uptake driven by the enhanced sequestration of free [Ca2+]m to maintain a transmembrane Ca2+ gradient and a charged ΔΨm that facilitated additional Ca2+ uptake (Figure 2). Unlike previous studies [14,33,34], NCLX was blocked under our experimental conditions, to prevent Ca2+ efflux during the repetitive CaCl2 additions; therefore, the net free ss[Ca2+]m in our study was determined by the balance between Ca2+ uptake and Ca2+ sequestration.

Notably, the CsA-induced buffering of mitochondrial Ca2+ resulted in greater Ca2+ uptake to attain a steady-state, as shown by the gradual decrease in ss[Ca2+]m with each added CaCl2 pulse (Figure 3). Insofar as Ca2+–Pi precipitation is a major mechanism for mitochondrial Ca2+ buffering, the sustained ss[Ca2+]m after each CaCl2 bolus indicated matrix Ca2+ storage, likely in the form of various inorganic Ca–Pi complexes [14]. The low and maintained ss[Ca2+]m during continuous matrix Ca2+ uptake is consistent with formation of these complexes. Although our study did not provide direct experimental evidence for CsA-induced matrix Ca–Pi complex formation, the continuous rise in estimated bound Ca2+:free Ca2+ ratio with each CaCl2 bolus as well as the ten-fold increase in mβCa clearly reflects a CsA effect on [Ca2+]m buffering capacity (Figure 3).

The protective effect of CsA in delaying mPTP opening has long been reported [28,31,32]. Our findings; however, provide the first direct evidence for a novel effect of CsA to enhance the capacity of mitochondria to sequester Ca2+ by which it obviates Ca2+-induced mPTP formation. Moreover, the effect of CsA in mediating greater matrix Ca2+ buffering explains the sustained free [Ca2+]m reported by Chalmers and Nicholls [14] and the CsA-induced inhibition of mitochondrial Ca2+ efflux observed in other prior studies [33,34].

#### *4.2. Underlying Mechanism of the CsA-Mediated [Ca2*+*]m Regulation*

It is well established that mitochondria are able to sequester large amounts of Ca2+, while maintaining free [Ca2+]m over a range of 0.1 and 10 μM depending on the Ca2+ load [14]; however, the mechanism and kinetics for this are unclear. Matrix Ca2+ buffering capacity is determined by: i) The quantity of Ca2+ that can be retained, and ii) the Ca2+ threshold level for release when Ca2+ exchangers are blocked or maximally operated [48]. The role of Pi as a physiological buffer in regulation of [Ca2+]m has been extensively studied [14,44,45,49]. The major mechanism of Pi-mediated Ca2+ sequestration in mitochondria is believed to be achieved by formation of amorphous Ca2+–Pi complexes in the matrix [48,50,51], which in turn maintain the free [Ca2+]m at a low level. Hence, sustained [Ca2+]m cyclically promotes more Ca2+ uptake via the MCU due to better preservation of both the Ca2+ gradient and ΔΨm.

Though Pi plays an essential role in matrix Ca2+ buffering, Pi has also been suggested to induce mPTP opening [52]. A recent study associated Ca2+–Pi precipitation with complex I inhibition and reduced ATP synthase rate during Ca2+ overload [53]. Another report demonstrated that increasing [Pi] decreased the mitochondrial Ca2+ loading capacity [14]. It was suggested that the mPTP-sensitizing effects of Pi was likely due to its effect in decreasing matrix-free Mg2+, an mPTP inhibitor [20]. In addition, formation of polyphosphate, a known inducer of mPTP, could be a factor in regulating the Ca2+ threshold for mPTP activation [54,55]. Interestingly, two prior studies [56,57] indicated that Pi is necessary for the inhibitory effect of CsA on mPTP opening. However, two other studies reported

that CsA inhibits mPTP opening even in the absence of Pi [58,59]. Conversely, in our study, the CsA-induced enhancement of matrix Ca2+ buffering was completely annulled when both mitochondria and the experimental medium were depleted of Pi (Figure 7). This loss of Ca2+ sequestration by CsA was reinstated when exogenous Pi was added just before activation of the mPTP (Figure 8). These observations provide the essential explanation for the requirement of Pi in the CsA-mediated MCBS and delay in mPTP opening.

The importance of mitochondrial matrix Ca2+ buffering via Pi is underscored by the studies of Wei et al. [44,45]. They reported that Pi modulates the total amount of Ca2+ uptake with smaller CaCl2 boluses, whereas Pi modulates Ca2+ buffering capacity with larger CaCl2 boluses. Since we had Pi in our experimental medium and the mitochondria were replete with exogenous Pi, the observation that CsA induced low ss[Ca2+]e and ss[Ca2+]m could be explained by the following: (i) CsA activates Pi-dependent matrix Ca2+ buffering potentially by maintaining the rate of Ca2+–Pi complex formation; and (ii) CsA may activate Pi transport processes (via H+/Pi transporter and/or phosphate carrier) that help to maintain both the IMM pHm and ΔΨm gradients. These processes would limit the increase in free [Ca2+]m, which in turn would contribute to more Ca2+ uptake and retention by increasing the electrochemical driving force for Ca2+ influx.

#### *4.3. CsA vs. ADP; As a Regulator of [Ca2*+*]m*

AdN are implicated as one of the multiple matrix factors responsible for sequestering Ca2+ by mitochondria [29,60–62]. AdN can potentiate mitochondrial Ca2+ buffering by maintaining high matrix Pi concentrations that can facilitate precipitation of AdN-Ca–Pi complexes, including, ATP-Mg2−/Pi2− and HADP2−/Pi2−, and thereby increase the Ca2+ threshold for mPTP opening [60,63]. In a study by Carafoli et al. [60], it was reported that mitochondrial Ca2+-buffering is proportional to mitochondrial ADP uptake. In our Pi-replete study, OMN+ADP had a relatively small effect on Ca2+ buffering compared to CsA, but it had a significantly larger effect than ADP or OMN alone (Figures 2, 3 and 5). A reasonable explanation could be that OMN, an ATP synthase (Complex V) inhibitor [64], could contribute towards augmenting the AdN pool and thus enhance matrix Ca2+ buffering. Consistent with our findings, a previous study also showed a greater CRC with a low-concentration of ADP with OMN compared to 10-fold larger concentration of ADP alone [30]. Thus, in agreement with Sokolova et al. [30], the observed high buffering capacity and expanded CRC with OMN+ADP is largely attributed to the ADP component of the matrix AdN pool. However, a previous study [62] reported that AdN also prevent mitochondrial Ca2+ influx by directly chelating Ca2+ by a Ca–ATP complexation [61]. Contrary to this observation, in our study, the direct effect of ADP on binding free Ca2+ was negligible, as assessed by adding ADP and CaCl2 together in mitochondria-free experimental buffer (Figure S4). Additionally, carboxyatractyloside-mediated inhibition of ADP uptake via adenine nucleotide translocase precluded matrix Ca2+ buffering and blunted the CRC by OMN+ADP or ADP alone (Figure S5). In this case, the extra-matrix ADP that accumulated did not chelate the Ca2+ added to the buffer. Altogether, these observations indicate that a direct sequestration of Ca2+ outside the mitochondria does not explain the effect of ADP alone or OMN+ADP on the enhanced CRC in our study.

A previous study [42] from our group proposed that the MCBS relies on at least two classes of Ca2+ buffers. The first class could represent classical Ca2+ buffers, including mostly metabolites (ATP, ADP, and Pi) and mobile proteins that bind a single Ca2+ ion at a single binding site. A second class of buffers could be associated with the formation of amorphous Ca2+ phosphates, which may be capable of binding multiple Ca2+ ions at a single site in a cooperative fashion [35,38,39,42]. Genge et al. [65] showed, in an in vitro study, that annexins, a diverse class of proteins, are required for Ca2+-phosphate nucleation. Additionally, many studies have suggested an AdN-dependent Ca2+-binding property of annexins [66]. Interestingly, mitochondria exposed to ADP alone or OMN+ADP retained their ability to maintain low ss[Ca2+]e and ss[Ca2+]m for an extended period of cumulative CaCl2 additions, and showed a higher Ca2+ threshold for mPTP opening without Pi compared to with Pi (Figure 7). This

extended delay in mPTP opening in the Pi-depleted state compared to the Pi-replete state reflects the ability of Pi to induce early mPTP opening under certain conditions [52]. In this case, the presence of Pi appears to counteract the ADP delay effect and induce a much earlier pore opening compared to the Pi-depleted state. The mechanisms for this AdN-mediated massive matrix Ca2+ loading capacity in the absence of exogenous Pi is unclear and needs to be further investigated. A plausible hypothesis could be that, in the absence of Pi, a significant Ca2+ loading capacity of AdN might be mediated via direct interaction with annexins. CsA, on the other hand, might function as a mediator that activates a Pi-dependent Ca2+ buffering system. Another possibility is that Cyp D, as a PPIase, reduces free phosphate levels in the matrix or blocks the Ca2+ binding property of annexins; this then would be relieved by CsA's effect to block Cyp D.

#### *4.4. Implication of CsA-Mediated Ca2*+ *Bu*ff*ering on Mitochondrial Bioenergetics*

Elevated [Ca2+]m over the nanomolar range is reported to increase NADH generation in part by stimulating Ca2+-sensitive dehydrogenases of the TCA cycle [67,68] and activating the F0F1-ATP synthase [69], thereby accelerating oxidative phosphorylation (OXPHOS). However, excess mitochondrial free Ca2+ can dissipate ΔΨm and impede OXPHOS. The IMM ΔΨm is the key factor in generating the proton motive force across the IMM; it is also one of the primary driving forces for Ca2+ uptake via the MCU [70] and triggers Ca2+ efflux via the NCLX [71,72]. Therefore, if mitochondria continue to take up Ca2+ under increased extra-matrix Ca2+ exposure, the Ca2+ would have to be buffered or ejected to prevent excess free [Ca2+]m accumulation that could dissipate ΔΨm and increase oxidation of NADH.

The stability of Ca2+–Pi precipitates inside the mitochondrial matrix largely depends on pHm [50]. It is also proposed that the matrix [Pi] depends on the pH gradient (e.g., a change in pH from 7 to 8 has been estimated to increase [Pi] by a factor of 1000 [14,50]). Thus, matrix alkaline conditions could facilitate Ca2+–Pi precipitation, whereas matrix acidification could lead to a destabilization of the Ca2+–Pi precipitate and so enhance matrix free Ca2+ levels [14]. Consequently, we correlated the changes in [Ca2+]m with indices of mitochondrial bioenergetics (ΔΨm, NADH, and pH) (Figure 5) to have a better understanding of the CsA-mediated MCBS. Mitochondria exposed to CsA before the repetitive CaCl2 boluses, exhibited robust mitochondrial Ca2+ uptake and rapid [Ca2+]m buffering while maintaining basal ΔΨm, NADH, and an alkalinized pHm until mPTP opened (Figure 5). Maintaining ΔΨm during excess Ca2+ uptake in the absence of functioning NCLX suggests a strong matrix buffering effect that is induced by CsA.

In Protocol B, when CsA was added just before the onset of pore opening, NADH and ΔΨm levels transiently increased but immediately returned to baseline with each added CaCl2 bolus. This transient depolarization and NADH oxidation with each addition of CaCl2 was not observed in Protocol A. The reason for this is unclear. Nonetheless, the observed transient oxidation of NADH helped to restore ΔΨm after Ca2+ induced transient depolarization before the next CaCl2 bolus (Figure 5D,E). The transient redox oxidation and ΔΨm depolarization suggest that the CsA added at the point just before mPTP opening activated MCBS more slowly compared to Protocol A. In addition, CsA maintained the pHm gradient during prolonged Ca2+ pulse challenges (Figure 5C). This finding also likely excludes a contribution of the mitochondrial calcium–hydrogen exchange (mCHE) to the Ca2+ extrusion in the absence of NaCl. We have recently reported that CsA obviates mCHE activity at low extra-matrix pH [19]. However, based on our current results, it is likely that CsA triggered an enhancement of mitochondrial Ca2+ buffering so that the resulting low [Ca2+]m and maintained ΔΨm and pHm accounted for the inactivity of mCHE.