Abstract
Background
Methods
Results
Conclusions
Introduction
Prevalence of frailty is higher among persons living with HIV (PLWH) than age-matched controls [1–3] and is associated with increased fall rates [4,5]. In addition to its skeletal effects, low 25-hydroxyvitamin D (25-OHD), particularly levels <20 ng/ml, has been associated with impaired muscle strength, function and balance in the general population, which has important implications for falls and fracture risk [6–8]. Vitamin D receptor (VDR) is expressed abundantly in skeletal muscle cells. Vitamin D (VitD) deficiency results in atrophy of these muscle fibres, fat infiltration and resultant fibrosis [8]. In a randomized, placebo-controlled study of older, mobility-limited women with moderately low 25-OHD levels, supplementation with 4,000 IU VitD3 resulted in a 30% increase in intramyonuclear VDR protein and a 10% increase in total (type I and II) muscle fibre size over a 4-month period [9].
Few studies have specifically evaluated impact of VitD supplementation on neuromuscular outcomes among PLWH [10]. A recent randomized clinical trial of 56 children and young adults living with HIV compared the effects of 7,000 IU of VitD daily versus placebo over 12 months and found a mild increase in test scores for fine and gross motor skills, but no change in other parameters. There are no published studies on the effect of VitD supplementation in older PLWH who may benefit most.
Provision of adequate calcium and VitD is the cornerstone of effective prevention of osteoporosis and its consequences, as VitD in particular has been shown to reduce bone loss and falls, and lower the risk of hip fracture in older individuals [11–13]. A comprehensive meta-analysis of randomized controlled trials found that among adults with baseline 25-OHD <10 ng/ml, VitD supplementation resulted in increased handgrip strength [14] and that improvements in muscle function were associated with increased lean body mass [15].
We have previously published the results of a randomized clinical trial of two doses of VitD3 repletion (3,000 IU versus 1,000 IU) on bone turnover and change in areal and volumetric bone mass and microarchitecture in minority postmenopausal women living with HIV [16]. We found changes in bone mineral density and bone microarchitecture did not differ between moderate and low VitD treatment groups. We now report on the muscle mass and muscle strength data for this study.
Methods
Study population
Postmenopausal African American or Hispanic women living with HIV between ages 40-70 were recruited from four sites in New York City (Columbia University Irving Medical Center, Mt Sinai St Luke's and Mt Sinai West, Bronx Lebanon Hospital Center, and Montefore Medical Center, New York, NY, USA). All participants had serum 25-OHD ≥10 ng/ml and ≤32 ng/ml and were on stable antiretroviral therapy (ART) for at least 2 years with 2 or more HIV RNA levels <400 copies/ ml within the past year prior to enrolment. Exclusion criteria included: metabolic bone diseases, renal insufficiency (serum creatinine >1.5 mg/dl or estimated glomerular filtration rate <45 ml/min/1.73 2 ), current use of glucocorticoids or hormone replacement therapy, current or past osteoporosis therapy, history of fragility fracture or T-score <-3.0, history of hypercalcaemia or calcium-containing kidney stones, and weight over 300 lbs.
Participants were randomized to receive 3,000 versus 1,000 IU cholecalciferol (vitamin D3) daily (Tishcon Corporation, Westbury, NY, USA) plus 500 mg calcium carbonate twice daily and counselled to take with food to facilitate absorption. Participants were randomized in a 1:1 ratio using permuted blocks stratified by screening serum 25-OHD (≤20 and >20 ng/ml). Sample size for the main study was based upon the primary bone mineral density outcomes which have been reported [16]. Adherence to VitD and calcium was monitored by counts of returned pills at each 3-month study visit. The Institutional Review Boards of all participating sites approved the study. Secondary outcomes include change in appendicular lean mass by dual-energy X-ray absorptiometry (DXA), muscle function and frailty measures from baseline to 12 months. Changes in plasma 25-OHD, parathyroid hormone (PTH) over 12 months were also examined in relation to the skeletal muscle measures. All DXA scans and muscle function measures were performed at Columbia University Irving Medical Center (CUIMC; New York, NY, USA) using a standardized protocol.
Lean body mass measures
Body composition was measured by DXA on a QDR 4500 bone densitometer (Hologic, Inc., Bedford, MA, USA) at CUIMC and analysed using established protocols. Height and weight were measured by Harpenden stadiometer and balance beam scale, respectively. Non-fat and non-bone tissue was defined as lean body mass.
Muscle function testing
Muscle functional testing was performed by trained staff under the supervision of a physician. Biodex System 4 Pro (Biodex Medical Systems, Shirley, NY, USA) was used to measure both isometric and isokinetic movements. The dominant knee was tested first, followed by the dominant elbow.
For isometric contractions, participants contracted against a constant 60-degree angle at the knee and a constant 30-degree angle at the elbow for 6 s. Participants alternated between flexion and extension, three repetitions each with a 30 s rest period between each. Data were collected to determine peak torque to body weight (PKTQ/BW).
For isokinetic contractions, participants performed flexion and extension movements at the knee and elbow for five repetitions at a speed of 60 degrees/s. This was followed by a rest period of 2.5 min. For the second isokinetic protocol, participants performed 25 repetitions of flexion and extension movements at the knee and elbow at a speed of 180 degrees/s. Data were collected to determine PKTQ/BW, average power, work to body weight (WK/BW), fatiguability, and time to peak torque. Normalization of the data to body weight allowed for a more meaningful comparison of measurements among participants.
Frailty was defined as the presence of ≥3 of the following criteria: unintentional weight loss, self-reported exhaustion, weakness, slow walking speed, and low physical activity, using the same cut points developed by Fried et al. [17] in the Cardiovascular Health Study (CHS) study.
Biomarker assays
Serum samples were stored at -70°C until batched analysis at the Irving Institute Biomarkers Core at Columbia University Medical Center. We measured 25-OHD2 and D3 by liquid chromatography tandem mass spectrometry, and the total reported as the sum of 25-OHD2 and D3; calibration standards for both 25-OHD2 and D3 were validated against National Institute of Standard and Technology standard, SRM972a. We also measured intact PTH by radioimmunoassay (Scantibodies, Santee, CA, USA), interleukin (IL)-6 (ELISA; R&D Systems, Minneapolis, MN, USA), soluble receptors of tumour necrosis factor (TNF)-α (soluble [s]TNFr-I and -II; ELISA; R&D Systems), and soluble CD14 (sCD14; ELISA; R&D Systems). Except for 25-OHD, biomarkers were measured in duplicate and values averaged for analysis.
Statistical analysis
A complete case approach was utilized for all lean mass, strength and frailty analyses in participants at 48 weeks (n=69). Stratified Wilcoxon rank-sum tests were used to test for distribution shifts between the treatment groups. Fisher's exact tests and Wilcoxon rank-sum tests were used to evaluate for differences between groups for categorical and continuous secondary outcomes, respectively. Wilcoxon signed-rank test was used to evaluate within treatment group change. All statistical tests were two-sided and interpreted at the 5% level of significance without adjustment for multiple comparisons. Analyses were performed using R 3.4.0.
Results
Between 2011 and 2016, we enrolled and randomized 85 participants who were included in the intent-to-treat analysis. Among all participants, 82 received at least one dose of the allocated intervention (41 in low VitD and 41 moderate VitD). Overall, 69 participants (31 in the low VitD and 38 in the moderate VitD groups) completed the 12-month study with complete muscle function and interpretable DXA data and were included in the complete-case analysis [16].
Baseline demographic, immunological and other parameters are summarized in Table 1 for the 69 participants in the complete-case analysis. Overall, the mean age was 56.6 years, 42% were African American, 26% Hispanic, median CD4+ T-cell count 738 cells/mm3 and 71% had HIV RNA≤50 copies/ml. Age, race/ethnicity, proportion with diabetes and HIV-specific parameters such as current/nadir CD4+ T-cell count, history of AIDS-defining illness, ART class and proportion with HIV RNA levels <50 copies/ml were similar between groups. However, at baseline, the moderate VitD group had lower BMI, total body fat and trunk fat (Table 1). Mean 25-OHD and PTH did not differ between treatment groups; 52.6% and 51.6% had VitD deficiency with 25-OHD <20 ng/ml in the moderate VitD and low VitD groups, respectively (P=0.934). Serum levels of inflammatory biomarkers (TNF-α, IL-6 and sCD14) did not differ between treatment groups.
Baseline characteristics of participants who received allocated intervention and completed 12-month study evaluation a
n=69. Data are mean ±
Change in 25-OHD and PTH
Serum levels of total 25-OHD were higher in the moderate than low VitD group at 6 months (33.1 ±10.6 versus 28.6 ±8.2 ng/ml, P=0.031) and 12 months (30.2 ±9.8 versus 24.8 ±8.0 ng/ml, P=0.014). Similarly, percentage change from baseline in serum 25-OHD levels were greater in the moderate than low VitD groups at 6 months (67 ±78% versus 45 ±44%, P=0.041) and 12 months (51 ±69% versus 28 ±42%, P=0.031). Overall, PTH levels decreased in both groups, but the decrease did not differ between moderate and low VitD groups (data not shown).
Change in lean mass by DXA at 12 months
At baseline, mean upper and lower extremity lean mass at the dominant side were similar between two the treatment groups (Table 1). Weight was relatively stable over the 12-month course of study, and change in weight did not differ between the low VitD versus moderate VitD groups, respectively: +0.31 kg versus -0.40 kg (P=0.46). Similarly, there was no significant within-subject change in lean mass in either the upper or lower extremity in either treatment group (Tables 2 and 3). Among participants with 25-OHD <20 ng/ml at baseline, there were no significant differences in lower extremity or upper extremity lean mass at baseline or after 12 months between low and moderate VitD treatment groups.
Upper extremity muscle mass and strength parameters at baseline and 12 months by treatment arm
Data are mean ±
Lower extremity muscle mass and strength parameters at baseline and 12 months by treatment arm
Data are mean ±
Change muscle strength at 12 months
Skeletal muscle strength parameters are shown for the dominant limb in the upper extremity in Table 2 and in the lower extremity in Table 3. At baseline, there were no significant differences between groups in isometric and isokinetic muscle strength in either the upper or lower extremities.
In the low VitD group, there were significant within-group increases in upper extremity isometric muscle strength, peak torque (extension) from baseline to 12 months, but no increases in other muscle groups. There were no apparent increases in isokinetic muscle strength (5 repetitions) from baseline to 12 months in the upper extremities (Table 2), but there were significant within-subject changes in peak torque (flexion), total work (flexion) and average power (flexion) in the lower extremities (Table 3). Similarly, there were no apparent increases in isokinetic muscle strength (25 repetitions) from baseline to 12 months in the upper extremities (Table 2), but significant within-subject change in total work (flexion) and trends for peak torque (flexion) and average power (flexion) in the lower extremities (Table 3).
In the moderate VitD group, there were no significant within-group increases in upper extremity isometric muscle strength. There were no apparent increases in isokinetic muscle strength (5 repetitions) from baseline to 12 months in either the upper extremities (Table 2), but there were significant within-subject change in peak torque (flexion), total work (flexion) and average power (flexion and extension) in the lower extremities (Table 3). There were no apparent increases in isokinetic muscle strength (25 repetitions) from baseline to 12 months in either upper or lower extremities (Tables 2 and 3).
Greater increase in muscle strength was apparent in the moderate versus low VitD group in lower extremity isokinetic strength, 5 repetitions, only for peak torque (flexion) (26.1 ±11.5% versus 16.3 ±6.4%, P<0.05; Figure 1). Adjustment for BMI attenuated the between-group difference (P=0.372). None of the other between-group comparisons in change in muscle strength (isometric, isokinetic 5 repetitions and isokinetic 25 repetitions) were statistically significant (data not shown).
Isokinetic strength in lower extremities a
Five repetitions: percentage increase in muscle strength over 12 months in women receiving low versus moderate vitamin D (VitD) supplementation. BW, body weight.
Change in functional measures after 12 months
At baseline, there were no significant differences between groups in grip strength, 4-metre walk speed or time to complete 10 chair stands. There were significant within-group improvements in chair stand time in both the moderate VitD (31.5 ±8.5 to 26.2 ±7.9 s, P<0.0001) and low VitD (29.6 ±9.7 to 24.6 ±7.3 s, P=0.009) after 12 months; however, there were no significant differences between treatment groups. There were no significant within-group improvements in any other muscle function test in either treatment group. Overall, there was a very low prevalence of frailty, with only 1/38 (3%) of the moderate VitD and 2/31 (6%) of the low VitD group meeting frailty criteria.
Discussion
In this study, we found that VitD supplementation at 3,000 IU daily increased mean total 25-OHD levels to ≥30 ng/ml in minority postmenopausal women living with HIV, with 53% achieving 25-OHD levels >30 ng/ml at 12 months. Improvements in muscle function were apparent in both groups receiving VitD (3,000 IU and 1,000 IU). Greater improvements were apparent in isokinetic muscle function for knee flexion (peak torque) with moderate (3,000 IU daily) versus low (1,000 IU daily) VitD supplementation, but did not remain significant after adjustment for BMI. Improvements in muscle strength were not associated with increase in muscle mass or weight.
Provision of adequate calcium and VitD is the cornerstone of effective prevention of osteoporosis and its consequences, as VitD in particular has been shown to reduce bone loss and falls and lower the risk of hip fracture in older individuals [11–13]. A comprehensive meta-analysis of randomized controlled trials in the general population found that among adults with baseline 25-OHD <10 ng/ml, VitD supplementation resulted in increased handgrip strength [14] and that improvements in muscle function were associated with increased lean body mass [15]. From existing studies of VitD supplementation in postmenopausal women, doses of 1,000 IU per day or greater have resulted in improvement in physical measures in randomized clinical trials. In a study of 70-90-year-old postmenopausal women (n=302), Zhu et al. [18] found that VitD 1,000 IU plus calcium citrate once daily for a year resulted in improved muscle strength and mobility as measured by strain gauge and get up and go for women in the lowest tertile of muscle function compared with placebo. In a study of 50-65-year-old postmenopausal women (n=160), Cangussu et al. [19] found that VitD 1,000 IU/day with calcium supplementation taken for 9 months improved chair rising test and maintained lean body mass by DXA in comparison to the placebo.
Several studies suggest that frailty is more common among PLWH than uninfected controls and that the presence of frailty is associated with future falls [5,20]. Prevalence of frailty using Fried's frailty phenotype was 11.3% among women aged 70-79 in the Women's Health and Aging Studies (WHAS) [21] and 11.0% among minority women aged 65-70 in the CHS [17]. In a prior study of postmenopausal Hispanic and African American women with HIV in New York City with a mean age of 58, we found a 11% prevalence of frailty using the same criteria [1].
Two recent studies evaluated risk factors for falls among PLWH in comparison to control. The first, found that among PLWH, smoking, higher number of medications and imbalance symptoms were positively associated with falls, whereas protease inhibitor use appeared to be protective [22]. In the second study, factors independently associated with falls included factors affecting cognition and balance such as age, marijuana use, number of central nervous system active agents, subjective cognitive complaints, depressive symptoms and neuropathy [20]. Neither study evaluated associations between falls and VitD levels. In this study sample of postmenopausal women with a mean age of 56, we had a much lower prevalence of frailty (4.3%) than expected and none of the participants had falls or fractures during the course of the study. The lower prevalence of frailty and lack of falls in our sample, may have accounted for the modest observed effect with the VitD supplementation.
The major focus of VitD supplementation in PLWH has been to address skeletal complications, such as bone loss and fractures. Although there has also been interest in assessing the effects of VitD supplementation on muscle function, immune modulation and prevention of HIV- and non-AIDS-related outcomes, only one randomized clinical study has evaluated the potential impact of VitD supplementation on neuromuscular outcomes in PLWH [10]. This study randomized 56 children and young adults living with HIV to receive 7,000 IU of VitD daily versus placebo over 12 months and measured a battery of neuromuscular motor skills including the Bruininks-Oseretsky test of motor performance, jump power and energy, and muscular force and strength. The authors observed a mild increase in Bruininks-Oseretsky test score (β=1.14; P=0.041), a measure of gross and fine motor skills, but no change was observed in other parameters. There are no studies of VitD supplementation and muscle function in older PLWH.
In our study, median 25-OHD levels were ≥30 ng/ml at both the 6- and 12-month time points in the moderate and low VitD treatment arm, with over 56% and 53% of women achieving levels >30 ng/ml at 6 and 12 months, respectively. We observed differences between treatment groups only in isokinetic knee flexion, but not in other strength parameters. However, these improvements were modest and were not significant after adjustment for between-group differences in BMI. Furthermore, without a true placebo group, we are limited in our interpretation of these changes, since there is a well-recognized ‘training effect’ with the Biodex and with muscle function tests.
The most comprehensive Biodex data in older adults come from a study of Flemish Caucasian adults (19-73 years; 381 men and 210 women) that evaluated baseline and 10-year change muscle strength using the Biodex Medical Systems 3 dynamometer [23,24]. Among women age 40-60, at baseline the mean isometric peak torque leg extension measure (122.2 ±25.8 nM) is very similar to our measure. Their isokinetic measures differ from ours and therefore are difficult to compare. Among women between ages 40-60, the percentage change in isometric peak torque leg extension strength was -1.09 ±2.11% per year and isokinetic isometric peak torque was -0.81 ±2.29% [23]. Decrease in strength was even more profound in women over age 60, with a mean change of -1.92 ±1.74% per year in isometric and -1.5 ±2.67% change in isokinetic leg extension strength [23]. The mean age of our study population was 56; therefore, it is possible that both our VitD supplementation regimens had a positive effect on muscle function and we would have been able to detect this difference if we had included a VitD placebo group.
Our study had several limitations. At the time the study was conceived, a large amount of observational data suggested that VitD supplementation was beneficial in multiple domains, and there were ethical concerns about randomizing severely VitD deficient postmenopausal women to placebo for 12 months. Therefore, for ethical reasons, we chose to compare a lower (1,000 IU/day) dose with a higher (3,000 IU/day) dose that was below the 4,000 IU set by the Institute of Medicine guidelines as the upper limit of daily VitD supplementation [25], rather than conducting a placebo-controlled trial. Also, our modest sample size and higher rate of discontinuation than anticipated may have limited our ability to detect more consistent benefits of therapy. The 25-OHD levels at baseline may not have been sufficiently low to be able to result in large improvement in muscle function with VitD supplementation. DXA does not distinguish between specific muscles in the arms and legs; therefore, correlations between appendicular lean mass and muscle strength may underestimate stronger correlations between specific muscle groups and strength. Another limitation is that there are no established cut points for Biodex that can be related to clinical outcomes, such as physical disability and functional impairment, unlike skeletal muscle mass and skeletal muscle index [26]; therefore, the clinical significance of Biodex findings are less clear.
In conclusion, our study found modest improvements in some parameters of lower extremity muscle function in both VitD treatment groups, without concomitant increase in lean mass. We believe that higher doses of VitD could be used in future studies, but the greatest impact is likely to occur in more compromised subgroups, such as in patients with frailty or older patients. Additional studies are required to determine the optimal dose of VitD to improve muscle strength, which groups of PLWH will benefit from supplementation, and whether supplementation reduces clinically meaningful outcomes such as decreasing falls and improving physical activity.
Footnotes
Acknowledgements
This work was supported by the National Institutes of Health through grants AI1065200 (ES), AI059884 (MTY), AR06199301 (AS) and the National Center for Advancing Translational Sciences, through grant number UL1TR001873. This content does not represent official views of the NIH.
The authors declare no competing interests.