The Association Between Hypovitaminosis D Status with Muscle Strength and Functional Capacity in a Brazilian Community-Dwelling Elderly Population: A Cross- Sectional Study

Background: Although there have been studies on the association between hypovitaminosis D status with muscle strength and functional capacity, there were few studies on Brazilian community-dwelling elderly population. Therefore, this study aims to assess the association of hypovitaminosis D with muscle strength and functional capacity in a sample Brazilian community-dwelling elderly population. Methods: An analytical, cross-sectional observational study with a convenience sample was conducted. The 25(OH)D levels were assessed using an enzyme-linked uorescent assay and classied according to the standardization of the Brazilian Society of Clinical Pathology/Laboratory Medicine. Hand grip strength (HGS) and isometric quadriceps strength (IQS) were measured using manual Jamar ® and Lafayette ® 01165 dynamometers, respectively. Functional capacity was evaluated using the Short Physical Performance Battery (SPPB). Multiple linear regression models were used to assess the association of hypovitaminosis D with the HGS, IQS, and SPPB score via the Enter method. The models were adjusted for age, sex, presence of pain, level of physical activity, and use of vitamin D supplementation. Results: A total of 150 elderly individuals (age: 67.18±5.33 years) were included. According to the 25(OH)D levels, 60.7% had hypovitaminosis D (insucient: 48%, decient: 12.7%), and 39.3% had sucient levels. No associations were found between hypovitaminosis D and the HGS (p=0.84), LogIQS (p=0.462), and SPPB score (p=0.617). In the regression model, the inuence of the vitamin D levels on functional capacity was not signicant, demonstrating no association between them. Conclusion: Hypovitaminosis D was not associated with decreased muscle strength and functional capacity in the sample Brazilian community-dwelling elderly population. Longitudinal studies with measurement the time of use and levels of vitamin D


Background
Hypovitaminosis D is a frequent nding in older populations. Approximately 1 billion individuals have low vitamin D levels, regardless of ethnicity, age group, and geographic location [1,2]. Depending on the population studied, the prevalence can reach up to 90%, being considered by many researchers as a public health problem. Approximately 50-70% of European older individuals, 20-100% of Americans, and one third of Taiwanese and Japanese older individuals had vitamin D levels lower than 20 ng/mL, indicating a de ciency [3,4]. In Brazil, despite being a country with a tropical climate, hypovitaminosis D occurs in several age groups mainly owing to a diet low in vitamin D, and affected individuals are rarely offered multivitamin than 250 body sites, including muscle tissue. Inconclusive evidence shows that vitamin D is directly related to the metabolism of muscle energy and regulation of consumption and dynamics of mitochondrial oxygen, besides playing an important role in the regulation of muscle tone and contraction [8].
Taken together, changes in the vitamin D levels may contribute to the deterioration of various systems; however, the relationship of hypovitaminosis D with muscle strength and functional capacity in older people populations has been a subject of con icting results [9][10][11][12]. Given the tropical climate of Brazil and the peculiarities inherent to the senescence of older people individuals living in a country with socio-economic and cultural inequities, we hypothesized that hypovitaminosis D would negatively in uence the muscle strength and functional capacity of older people individuals. Thus, the aim of this study was to examine the association of hypovitaminosis D (insu ciency and de ciency) with muscle strength and functional capacity in a sample Brazilian community-dwelling older people population.

Study design and participants
This study was an analytical observational study with a cross-sectional design. A convenience sample of 150 Brazilian community-dwelling older people individuals of both sexes, aged 60 years or older, and recruited from August 2019 to February 2020 participated in the study. The exclusion criteria were as follows: cognitive decline detectable on the Mini-Mental State Examination [13], neurological sequelae, acute diseases, neoplasms, gastrectomy, use of prosthesis, use of drugs that interfere with the vitamin D levels, sequelae of diseases that could cause decreased muscle strength of the hands and lower limbs, severe sequelae of fracture, and previous orthopedic surgeries.

Measurements Sociodemographic and clinical variables
To characterize the sample, we asked all participants to answer a clinical-sociodemographic questionnaire with items on age, sex, race, origin, marital status, schooling, profession, main diagnosis, presence of comorbidities, use of medications and supplements, and anthropometric data.

Laboratory measurements
The levels of 25(OH)D were measured using an enzyme-linked uorescent assay. The tests were conducted in the laboratory of SARAH Network of Rehabilitation Hospitals. The reference levels were determined according to the standardization of the Brazilian Society of Clinical Pathology/Laboratory Medicine (SBPC/ML) for individuals aged 60 years or older: 30-100 ng/mL, indicating vitamin D su ciency; 20-29.9 ng/mL, indicating insu ciency; and below 20 ng/mL, indicating de ciency [5].

Muscle strength
Muscle strength was evaluated on the basis of hand grip strength (HGS) and isometric quadriceps strength (IQS). The Jamar ® dynamometer was used to assess the HGS, as recommended by the American Society of Hand Therapist (ASHT) [14]. The Lafayette ® model 01165 manual dynamometer was used to assess the IQS, following the recommendations standardized by Bohannon (2012) [15]. In both parameter evaluations, three repetitions were performed with the dominant limb, with a 1-minute rest interval between each measurement; subsequently, the arithmetic mean was calculated [16,17].

Functional capacity
Functional capacity was measured using the Short Physical Performance Battery (SPPB) adapted and validated for Brazilian older people individuals [18].

Statistical analysis
The Shapiro-Wilk test was used to evaluate the normality of the data. The quantitative descriptive data of the sample were summarized by measures of central tendency (medians and interquartile interval dispersions). The categorical variables of the study were described as absolute frequencies and percentages and the quantitative variables as medians ( rst quartile; third quartile) because the variables did not have a normal distribution as veri ed in the normality test.
As this study was an exploratory study conducted with a convenience sample, we initially tested the effect of the use of vitamin D and use of supplements on the study-dependent variables (HGS, IQS, and SPPB score) and vitamin D status to assess whether these variables in uenced the outcome variables. The tests used were the Mann-Whitney (HGS and IQS), Pearson's exact chi-square (SPPB score), and asymptotic Pearson's chi-square tests (vitamin D status).
Multiple linear regression models were used to assess the association of the vitamin D levels with muscle strength (HGS and IQS) and functional capacity through the Enter method. The models were adjusted for age, sex, presence of pain, level of physical activity, and use of vitamin D supplementation. Since the IQS did not present a normal distribution, we decided to use the logarithm of the IQS (LogIQS).
The coe cient of multiple determination (R 2 ) was used to quantify the strength of the association between muscle strength and the independent variables. The F-test was used to determine the statistical signi cance at p-values of <0.05. Regarding the assumptions of the model, the presence of multicollinearity was taken into account when the variance in ation factor was >10, and the tolerance was <0.2; homoscedasticity was veri ed by observing the graph of the predicted values and observed values and the normality of the nonstandardized residue. For all analyses, the SPSS version 23.0 software for Windows was used.
For all analyses, the signi cance level was set at 0.05, and the SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA) was used.
Interestingly, the participants who were using supplements (p < 0.0001) and vitamin D (p < 0.013) presented signi cantly lower HGS values in both analyses. Meanwhile, there was no signi cant difference in the IQS and SPPB score between the participants who used and who did not use supplements and vitamin D (p ≥ 0.05) ( Table 2).
The multiple regression analysis showed that the vitamin D levels were not associated with the HGS and IQS.
The in uence of the vitamin D levels on the HGS and LogIQS lost its signi cance after the inclusion of the adjustment variables age, sex, presence of pain, level of physical activity, and use of supplements (Tables 3   and 4).
Regarding functional capacity, 2% had an SPPB score of ≤ 6, 15.3% between 7 and 9, and 82.7% between 10 and 12 (good). In the regression model, the in uence of the vitamin D level on functional capacity was not signi cant.

Discussion
This study conducted in a sample of 150 Brazilian community-dwelling older people individuals showed that the prevalence of hypovitaminosis D was high (60.7%); in contrast, there was no association of hypovitaminosis D with muscle strength and functional capacity. This nding may be attributed to the fact that the sample included older people individuals with lower active age groups and few comorbidities, and the majority was practicing physical activity.
The high prevalence of hypovitaminosis D in this sample corroborates previously reported ndings. Recently, a systematic review and meta-analysis of 72 studies on the prevalence of hypovitaminosis D in Brazil reported a rate of insu ciency of 45.85% and a rate of de ciency of 41.53%. The study mentioned included all age groups; however, it highlighted a greater presence of hypovitaminosis D among the older people group In recent decades, research has con rmed the important role of vitamin D in several outcomes, especially after the discovery of its activity on various organs and tissues [7]. Its relationship with the muscular system was more noticeable after the discovery of the VDRs in muscle tissue in the 1980s [19]. However, to date, the evidence on the in uence of vitamin D de ciency on the muscle strength and functional capacity of older people individuals is still inconclusive. Aspell et al. (2019) evaluated 4,157 individuals with a mean age of 69.8 ± 6.9 years and reported that vitamin D de ciency was associated with decreased muscle strength among English community-dwelling older people individuals [20]. The value adopted to verify the vitamin de ciency used in this study was 30 nmol/L, which corresponds to 12 ng/mL. In our study, the recommendations of the SBPC/ML established levels below 30 ng/mL to indicate insu ciency and below 20 vitamin D de ciency hampers a comparative analysis between the two studies [21].
In 2018, Mendes et al. evaluated 1,425 older people individuals (mean age: 75.4 ± 7.5 years in women and 74.2 ± 6.7 years in men) and found a signi cant association between vitamin D de ciency and decreased gait speed and HGS, especially in men (41.5% of the total sample) [22]. Of the participants of our study, only 28% were men, with a much smaller age group (67.18 ± 5.33 years), which could explain, at least in part, the con icting results. It is already established that men have greater muscle strength than women in all age groups and that the higher the age group, the lower the muscle strength and functional capacity [23]. Verlaan et al. (2018) conducted a multicenter study of 380 community-dwelling older people individuals aged over 65 years from 6 European countries (Belgium, Germany, Ireland, Italy, Sweden, and the United Kingdom) [24].
They found that older people individuals with vitamin D de ciency also presented decreased muscle mass (on DXA), strength (HGS), and functional performance in the SPPB (4-9 points). In our study, despite low schooling and living in a developing country, such as Brazil, 82.7% of the older people participants displayed good functional performance; further, 50.6% practiced regular physical activity, with few comorbidities, and were often exposed to sunlight. This differentiated pro le of the Brazilian older people individuals may have contributed to the results found.
Notably, some studies have demonstrated similar results: not nding an association between hypovitaminosis D and decreased muscle strength and functional capacity. Mathei et al. (2013) assessed the relationship between the levels of 25(OH)D, HGS, and gait speed and balance test ndings in 367 octogenarians. In this sample, only 12.8% presented vitamin D levels of 30 ng/mL or more [11]. Interestingly, there was no association found between hypovitaminosis D and the variables studied, although it was expected that the older age group would present worse results, which did not occur. Verreault et al. (2002) followed a cohort of 628 women (age: ≤65 years) for 3 years, performing evaluations every 6 months. The sample was divided into three groups according to the levels of vitamin D (≤ 10 ng/mL, 10-20 ng/mL, and ≥ 21 ng/mL) at baseline. Their analysis showed that the decline in strength (HGS) and muscle function (SPPB score) was similar in the three groups, regardless of the initial level of vitamin D [25]. Annweiler et al. (2009) evaluated the HGS, IQS, renal function, and parathyroid hormone (PTH) and calcium levels in 440 older people women with a mean age of 80.1 ± 3.5 years [26]. No association was found between vitamin D de ciency and decreased muscle strength. Advanced age and a higher number of chronic degenerative diseases were associated with greater decreases in muscle strength. The association of muscle weakness with increasing age is already well established in the literature and was one of the ndings.
A possible physiological explanation for the lack of association of vitamin D de ciency with muscle strength and functional capacity in the older people population found in this study could be the decrease in the number of VDRs in the muscle tissue. Currently, the exact mechanism of the relationship between receptors and vitamin D de ciency is not yet established, that is, whether this reduction is caused by vitamin D de ciency itself (negative regulation of the receptor) or if by another factor inherent to aging [27].
The complex interaction between muscle strength and vitamin D, especially in older people individuals, is still contradictory. The existence of possible confounding factors, such as the level of PTH, presence of kidney diseases, comorbidities, use of medications or supplements, and practice of physical activity, is not always controlled in studies, which may have contributed to the reporting of divergent results. Further, the large methodological variability, especially between the forms of measurement and classi cation of 25(OH)D, and diversity of assessments for muscle strength and functional capacity limit the comparison of data and may explain, at least in part, the large disparity of the ndings. Conversely, the heterogeneity of the aging process, senility, clinical and socio-cultural factors, and life habits could also interfere in muscle and functional performance and should also then be considered.
In this study, sex (female), increased age, presence of pain, and use of supplements were more related to decreased HGS than the serum level of 25(OH)D. Both sex and age are factors that explain the decrease in muscle strength, that is, men have higher HGS than women, and muscle strength decreases with age [23].
Our study had an exploratory design; however, it is important to highlight that we analyzed not only the serum levels of vitamin D and the functionalities but also the use of vitamin supplements. Herein, we found that the older people participants who were using some type of supplementation had lower HGS, which may suggest that they were experiencing functional decline and thus had the need for supplementation.
There was no association between hypovitaminosis D and the IQS; increased age, sex, presence of pain, and physical activity were most related to this variable. The participants of this study were healthy communitydwelling older people individuals, of whom 50.6% practiced regular physical activity, and 82.7% had a good functional status. The performance of physical activity is considered a premise to reduce the physiological losses inherent to aging; it is widely studied and has been proven to be e cient in maintaining functional capacity and muscle strength [28]. The presence of pain could be a source of bias for the performance of muscle contraction required in the IQS assessment. In our sample, the older people participants who complained of pain in some joint presented lower quadriceps strength. In a systematic review with metaanalysis, Latey et al. (2017) reported that the intensity, frequency, and duration of pain are associated with muscle weakness [29].
Our study used the SPPB for the evaluation of functional capacity. Most of the older people participants presented good performance in the SPPB, which resulted in a low variability in this variable. Previous studies have reported that life habits, such as the practice of physical activity and good diet, can in uence the functional performance of older people individuals; however, the in uence of the vitamin D levels on these outcomes needs further investigation in future studies [24,26].
This study has several limitations. First, as a cross-sectional study, which has an inherent limitation of not allowing the establishment of a cause-effect relationship. Therefore, the ndings must be analyzed with caution. Second, the medications and supplements taken by the participants were not qualitatively assessed.
Last, we did not evaluate the diet or time of sun exposure, which are factors that may in uence the level of vitamin D.

Conclusions
In conclusion, among the healthy Brazilian community-dwelling older people participants with good functional performance, there was no association found between hypovitaminosis D and the HGS, IQS, and

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding
This study did not receive funding from agencies in the public, commercial, or non-pro t sectors.
Ethics approval and consent to participate All the experimente protocol for involving humans was in accordance to guidelines of national, international, institutional, and Declaration of Helsinki in the manuscript. All participants provided written informed consent. The study was approved by the Ethics Committees of the SARAH Network of Rehabilitation Hospitals and the Federal University of Minas Gerais, under the CAAE numbers 06716919.5.0000.0022 and 06716919.5.3001.5149, respectively. Data were anonymized and de-identi able with no personal information, with careful protection on con dentiality.    Table 4 Results of the logFIQ multivariate linear regression analysis.