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nutrients
Article
Vitamin D Supplementation Improves Quality of Life
and Physical Performance in Osteoarthritis Patients
Pacharee Manoy 1, Pongsak Yuktanandana 2, Aree Tanavalee 2, Wilai Anomasiri 3,
Srihatach Ngarmukos 2, Thanathep Tanpowpong 2and Sittisak Honsawek 2 ,3 ,*ID
1Program in Medical Sciences, Faculty of Medicine, Chulalongkorn University,
King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 1873 Rama IV Road, Pathumwan,
Bangkok 10330, Thailand; pachareeman@gmail.com
2Vinai Parkpian Orthopaedic Research Center, Department of Orthopaedics, Faculty of Medicine,
Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society,
1873 Rama IV Road, Pathumwan, Bangkok 10330, Thailand; ypongsak@gmail.com (P.Y.);
areetana@hotmail.com (A.T.); srihatach@hotmail.com (S.N.); thanathep1@yahoo.com (T.T.)
3Department of Biochemistry, Faculty of Medicine, Chulalongkorn University,
King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 1873 Rama IV Road, Pathumwan,
Bangkok 10330, Thailand; awilaiano@gmail.com
*Correspondence: sittisak.h@chula.ac.th; Tel.: +662-256-4482
Received: 3 May 2017; Accepted: 22 July 2017; Published: 26 July 2017
Abstract:
(1) Background: Lower levels of serum 25-hydroxyvitamin D (25(OH)D) are common
in osteoarthritis (OA) patients. However, the effect of vitamin D supplementation on muscle
strength and physical performance remains unclear. This study will investigate the effects of
vitamin D
2
supplementation on muscle strength and physical performance in knee OA patients;
(2) Methods: One hundred and seventy-five primary knee OA patients with low levels of serum
25(OH)D (<30 ng/mL) received 40,000 IU vitamin D
2
(ergocalciferol) per week for six months. Body
composition, muscle strength, physical performance, serum 25(OH)D level, leptin, interlukin-6 (IL-6),
parathyroid hormone (PTH), protein carbonyl, and metabolic profile were analyzed; (3) Results:
Baseline mean serum 25(OH)D levels in knee OA patients was 20.73 ng/mL. Regarding baseline
vitamin D status, 58.90% of patients had vitamin D insufficiency, and 41.10% had vitamin D deficiency.
After vitamin D
2
supplementation for six months, mean serum 25(OH)D level was 32.14 ng/mL. For
post-supplementation vitamin D status, 57.10% of patients had vitamin D sufficiency and 42.90% had
vitamin D insufficiency. From baseline to six months, there was a significant increase in mean serum
25(OH)D level (p< 0.001), while mean LDL cholesterol (p= 0.001), protein carbonyl (p= 0.04), and
PTH (p= 0.005) all significantly decreased. Patient quality of life (SF-12) and pain (visual analog scale,
VAS) both improved significantly from baseline to the six-month time point (p= 0.005 and p= 0.002,
respectively). Knee OA patients demonstrated significant improvement grip strength and physical
performance measurements after vitamin D
2
supplementation (p< 0.05); (4) Conclusions: Vitamin D
2
supplementation for six months reduced oxidative protein damage, decreased pain (VAS), improved
quality of life, and improved grip strength and physical performance in osteoarthritis patients.
Keywords: vitamin D2supplementation; osteoarthritis; muscle strength; physical performance
1. Introduction
Osteoarthritis (OA) is the most common cause of musculoskeletal disability and pain worldwide.
OA is characterized by the degradation of articular cartilage, including changes in subchondral bone,
osteophyte formation, joint space narrowing, and synovial inflammation [
1
]. Symptoms of disease
include joint pain, knee muscle wasting, and decreased range of motion, all of which lead to severe
Nutrients 2017,9, 799; doi:10.3390/nu9080799 www.mdpi.com/journal/nutrients
Nutrients 2017,9, 799 2 of 13
pain and disability in later life [
2
]. There are many risk factors that lead to early structural changes of
the knee among healthy individuals. Vitamin D deficiency may play a role in the pathogenesis of OA
on a clinical level [
3
]. Vitamin D deficiency has been associated with poor physical performance in
the elderly [
4
], and 63% of primary knee OA patients were found to have low vitamin D status [
5
].
Accordingly, lower levels of 25-hydroxyvitamin D (25(OH)D) were associated with greater knee pain,
increased progression of radiographic OA [6], and poor quadriceps function [7].
Vitamin D supplementation is an alternative treatment in elderly people who are at greater risk
of vitamin D deficiency and tend to have poor physical function. Several studies have reported that
vitamin D supplementation increases muscle strength, improves physical function, and decreases risk
of falls among older people with low level of serum vitamin D [
8
–
10
]. However, other previous studies
reported that vitamin D supplementation did not improve muscle strength or physical function [
11
–
13
].
Using the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index and visual
analog scale (VAS) assessment, the effects of vitamin D supplementation were reported to decrease
pain and improve knee function in OA patients [
5
]. In contrast, another previous study reported no
significant positive effect of vitamin D supplementation on the prevention of tibial cartilage loss or
improvement in WOMAC knee pain [14].
Although vitamin D
2
(ergocalciferol) and vitamin D
3
(cholecalciferol) are available over the
counter as dietary supplements and do not require a prescription, ergocalciferol is the only therapeutic
agent that is a first-line drug (category A) for vitamin D deficiency used in the hospitals and
public health sectors in Thailand. Given this disparity in the previous finding regarding vitamin D
supplementation in Thailand, vitamin D
2
(ergocalciferol) was used in this study for the investigation
of the role of vitamin D supplementation on muscle strength and physical performance in knee OA
patients with vitamin D insufficiency and deficiency. The secondary objective of this study was to assess
the possible benefits of vitamin D supplementation on metabolic risk factors, levels of inflammation,
adipokine, and oxidative stress.
2. Materials and Methods
2.1. Study Design and Participants
This controlled before–after study was conducted at the outpatient clinic of the Department of
Orthopedics at King Chulalongkorn Memorial Hospital during a February–December 2015 study
period. Two hundred and thirty-eight patients with knee OA agreed to participate. All had knee
OA based on the criteria of American College of Rheumatology classification. The inclusion criteria
were that the participants had symptomatic knee OA (Kellgren–Lawrence grading of
≤
2) and low
vitamin D status (25(OH)D < 30 ng/mL). The diagnosis of osteoarthritis is based primarily on patient
history, physical examination, and radiographic findings. Exclusion criteria included history of
knee surgery, primary hyperparathyroidism, rheumatoid or other inflammatory arthritis (i.e., septic
arthritis, gout), neurological condition (i.e., Parkinson’s disease, previous stroke), or inability to
perform physical activity.
One hundred and ninety-one patients met the study criteria and were included. Sixteen of
the included patients were not included in the final analysis for the following reasons: 13 patients
were lost of follow-up, two sustained fracture (hip and lower leg–one each), and one patient had
knee arthroscopy. A total of 175 knee OA patients completed the study protocol and were analyzed
(Figure 1).
The study protocol was approved by the Institutional Review Board of the Faculty of Medicine at
Chulalongkorn University (IRB approval No. 512/57). Written informed consent was obtained from
all participants prior to their participation in the study.
Nutrients 2017,9, 799 3 of 13
Nutrients 2017, 9, 799 3 of 13
Figure 1. Flowchart of the study protocol.
2.2. Interventions
The Endocrine Society guidelines suggest that 50,000 IU of vitamin D2 taken once a week for
eight weeks is necessary to achieve the levels of serum 25(OH)D consistently above 30 ng/mL in
adults [15]. In Thailand, the only available form of vitamin D2 (egocalciferol) is in the form of 20,000
IU/capsule. Therefore, each subject was asked to take 40,000 IU of vitamin D2 (two capsules of 20,000
IU ergocalciferol; the British Dispensary, Bangkok, Thailand) per week for six months in order to
evaluate the effect of vitamin D supplementation on muscle function and biochemical markers.
2.3. General Information
All participants were evaluated for knee pain using WOMAC and VAS evaluation instruments.
VAS score is based on a 0–10 point scale, with a higher score indicating a higher level of pain. The
participants were asked to put a mark on the line indicating their pain intensity at the present time
in response to the following question: “If “0” is “no pain” and “10” is “the worst pain”, where is your
average pain intensity now on the visual analog score (VAS)?” [16]. Total WOMAC score represented
the sum of three subscales, including pain, stiffness, and physical function. A higher WOMAC score
indicates worse pain, more stiffness, and increased functional limitations. A Thai version of the Short-
Form Health Survey (SF-12) evaluated health-related quality of life, including physical health
composite score (PCS) and mental health composite score (MCS), both of which range from 0 to 100,
with a higher score indicating better quality of life and well-being. Physical activity was evaluated
using the Thai version of the Physical Activity Questionnaire for Elderly Japanese (PAQ-EJ). PAQ-EJ
physical activity measurements mirror patterns of daily activity among elderly Thai and other elderly
Asian people [17]. PAQ-EJ scores were converted to metabolic equivalent of task (MET) hours per
week (MET h/week) [18].
2.4. Anthropometric and Body Composition Measurements
Height, weight, and waist circumference (WC) were determined using standard measurement
techniques. Body mass index (BMI) was calculated by dividing weight (kg) by the square of height
(m2). Appendicular skeletal muscle mass (ASM), percentage of total fat mass, fat mass (FM), and fat-
Figure 1. Flowchart of the study protocol.
2.2. Interventions
The Endocrine Society guidelines suggest that 50,000 IU of vitamin D
2
taken once a week for
eight weeks is necessary to achieve the levels of serum 25(OH)D consistently above 30 ng/mL in
adults [
15
]. In Thailand, the only available form of vitamin D
2
(egocalciferol) is in the form of
20,000 IU/capsule. Therefore, each subject was asked to take 40,000 IU of vitamin D
2
(two capsules of
20,000 IU ergocalciferol; the British Dispensary, Bangkok, Thailand) per week for six months in order
to evaluate the effect of vitamin D supplementation on muscle function and biochemical markers.
2.3. General Information
All participants were evaluated for knee pain using WOMAC and VAS evaluation instruments. VAS
score is based on a 0–10 point scale, with a higher score indicating a higher level of pain. The participants
were asked to put a mark on the line indicating their pain intensity at the present time in response to the
following question: “If “0” is “no pain” and “10” is “the worst pain”, where is your average pain intensity
now on the visual analog score (VAS)?” [
16
]. Total WOMAC score represented the sum of three subscales,
including pain, stiffness, and physical function. A higher WOMAC score indicates worse pain, more
stiffness, and increased functional limitations. A Thai version of the Short-Form Health Survey (SF-12)
evaluated health-related quality of life, including physical health composite score (PCS) and mental
health composite score (MCS), both of which range from 0 to 100, with a higher score indicating better
quality of life and well-being. Physical activity was evaluated using the Thai version of the Physical
Activity Questionnaire for Elderly Japanese (PAQ-EJ). PAQ-EJ physical activity measurements mirror
patterns of daily activity among elderly Thai and other elderly Asian people [
17
]. PAQ-EJ scores were
converted to metabolic equivalent of task (MET) hours per week (MET h/week) [18].
2.4. Anthropometric and Body Composition Measurements
Height, weight, and waist circumference (WC) were determined using standard measurement
techniques. Body mass index (BMI) was calculated by dividing weight (kg) by the square of height
(m
2
). Appendicular skeletal muscle mass (ASM), percentage of total fat mass, fat mass (FM), and
fat-free mass (FFM) were assessed using bioelectrical impedance analysis (BIA) (BC-418 Segmental
Body Composition Analyzer; Tanita Corporation, Tokyo, Japan). ASM was estimated as the sum of the
Nutrients 2017,9, 799 4 of 13
skeletal muscle mass of the arms and legs in kilograms. The appendicular skeletal muscle mass index
(ASMI) was calculated as ASM divided by squared height. Skeletal muscle index (SMI) was calculated
as percentage of appendicular skeletal muscle mass (ASM) divided by body weight (%).
2.5. Muscle Strength and Physical Performance
At baseline, three months, and six months, muscle strength and physical performance were
measured by physical therapists. Grip strength was assessed by grip strength dynamometer (Takei
Scientific Instruments Co. Ltd., Tokyo, Japan) (kilograms) [
19
]. Knee extension force was measured by
a handheld MicroFET 2 dynamometer (Hoggan Scientific LLC, Salt Lake City, UT, USA) (Newtons).
The participant sat on the treatment table with knees flexed 90
◦
and the dynamometer was applied
to the anterior part of the leg, 5 cm above the transmalleolar axis and perpendicular to the tibial crest.
The participant raised their lower legs up and held against a maximum persistent force position (5 s)
applied by a physical therapist [
20
]. Four tests were used to evaluate physical performance. The first test
was the 4-m gait speed test, which measures the time needed to walk four meters, calculated as meters
per second [
19
]. The second test was the Timed Up and Go Test (TUGT), which measured the time
needed to stand up from a chair, walk three meters, and return to the chair and sit down (seconds) [
21
].
The third test was the five times sit-to-stand test (STS), which recorded the time needed to perform five
repeated chair stands without the use of arms (seconds) [
22
]. The last of the four tests was the six-minute
walk test (6MWT), which measured the distance a patient could walk in six minutes (in meters) [21].
2.6. Biochemical Analysis
At baseline and six months, fasting early-morning venous blood was collected and centrifuged,
with serum and plasma samples stored at
−
70
◦
C until use. Fasting blood glucose (FBG), lipid
profile, calcium, phosphorus, and high-sensitivity C-reactive protein (hs-CRP) were measured
using an autoanalyzer (Architech 16,000 Analyzer, Abbott Diagnostics, Irving, TX, USA). Serum
levels of leptin and interlukin-6 (IL-6) were determined by enzyme-linked immunosorbent
assay using kits from R&D Systems, Minneapolis, MN, USA and BioLegend, San Diego,
CA, USA, respectively. Plasma level of protein carbonyl was assessed by spectrophotometer,
according to the method of Castegna et al., 2003 [
23
] Serum 25(OH)D level was measured by
chemiluminescent immunoassay (DiaSorin, Inc., Stillwater, MN, USA). PTH and insulin were
determined by electrochemiluminescence method (Roche Diagnostics GmbH, Mannheim, Germany).
Insulin resistance was calculated using homeostasis model assessment (HOMA-IR) using the following
formula:
fasting serum insulin (µU/mL) ×fasting plasma glucose (mg/dL)/405
. Vitamin D deficiency
was defined as <20 ng/mL, vitamin D insufficiency as
20 -< 30 ng/mL
, and vitamin D sufficiency
as ≥30 ng/mL.
2.7. Statistical Analysis
Data were analyzed using SPSS Statistics version 22 (SPSS, Inc., Chicago, IL, USA). Comparison
of baseline vs. post-vitamin D
2
supplementation data was performed by paired t-test. One-way
repeated-measurement ANOVA was used to test the time differences in muscle strength and physical
performance. Correlation between variables was tested by Spearman’s rank correlation coefficient
(r). Data are summarized as mean
±
standard error of the mean (SEM). A p-value less than 0.05 for
differences and correlations was considered to be statistically significant.
3. Results
3.1. Effects on Body Composition, Pain, Quality of Life and Physical Activity
A total of 175 participants (158 females and 17 males) with a mean age of 64.58
±
0.55 years. After
vitamin D
2
supplementation, weight, percent of fat, fat mass, and visceral fat were all significantly
decreased, when compared to baseline levels (p< 0.05) (Table 1).
Nutrients 2017,9, 799 5 of 13
WOMAC and PAQ-EJ scores did not change significantly between baseline and six months.
However, VAS decreased significantly after treatment (p= 0.002) and the PCS of SF-12 improved
significantly after supplementation treatment (p= 0.005).
Table 1. Demographic data before and after vitamin D2supplementation in six months.
Vitamin D2Supplementation (n= 175)
p-Value
Mean ±SEM Mean Difference
(95% CI)
Baseline 6 Months
Gender (F/M) 158:17 158:17
Age (years) 64.58 ±0.55 64.58 ±0.55
Body composition
Waist circumference (cm) 87.87 ±0.73 87.82 ±0.71 −0.05 (−0.66 to 0.56) 0.87
Weight (kg) 62.38 ±0.89 61.70 ±0.88 −0.68 (−1.27 to −0.09) 0.02
BMI (kg/m2)25.63 ±0.30 25.41 ±0.30 −0.22 (−0.48 to 0.04) 0.09
Percentage of fat (%) 35.42 ±0.52 33.28 ±0.52 −2.14 (−2.80 to −1.47) <0.001
Fat mass (kg) 22.66 ±0.59 20.93 ±0.54 −1.72 (−2.30 to −1.14) <0.001
Visceral fat rating 9.46 ±0.29 9.03 ±0.24 −0.43 (−0.72 to −0.13) 0.004
ASM (kg) 17.58 ±0.28 17.50 ±0.27 −0.08 (−0.20 to 0.03) 0.15
ASMI (kg/m2)7.20 ±0.08 7.18 ±0.08 −0.03 (−0.08 to 0.01) 0.18
SMI (%) 28.37 ±0.27 28.52 ±0.25 0.14 (−0.15 to 0.44) 0.33
VAS (0–10) 3.96 ±0.17 3.44 ±0.17 −0.51 (−0.83 to −0.19) 0.002
WOMAC
Pain (0–10) 2.45 ±0.15 2.59 ±0.15 0.14 (−0.15 to 0.44) 0.33
Stiffness (0–10) 2.56 ±0.18 2.26 ±0.16 −0.29 (−0.62 to 0.03) 0.08
Physical disability (0–10) 2.90 ±0.15 2.76 ±0.15 −0.14 (−0.41 to 0.13) 0.31
Total score (0–10) 2.80 ±0.13 2.78 ±0.13 −0.01 (−0.08 to 0.06) 0.75
SF-12
PCS (0–100) 38.26 ±0.65 40.24 ±0.67 1.98 (0.60 to 3.36) 0.005
MCS (0–100) 50.00 ±0.70 49.57 ±0.66 −0.42 (−1.82 to 0.97) 0.54
Physical activity
PAQ-EJ (MET hours/week) 52.28 ±2.83 53.29 ±3.08 1.00 (−5.11 to 7.13) 0.74
Metabolic risk factors
FBG (mg/dL) 98.06 ±1.30 98.49 ±1.56 0.42 (−2.26 to 3.12) 0.75
Insulin (µU/mL) 5.32 ±0.41 5.99 ±0.41 0.66 (−0.20 to 1.53) 0.13
HOMA-IR 1.34 ±0.11 1.55 ±0.13 0.20 (−0.06 to 0.46) 0.13
Total cholesterol (mg/dL) 211.94 ±2.93 212.90 ±3.14 0.95 (−4.35 to 6.26) 0.72
HDL cholesterol (mg/dL) 55.30 ±1.00 57.40 ±1.28 2.09 (−0.03 to 4.23) 0.05
Triglycerides (mg/dL) 126.34 ±4.16 123.70 ±4.63 −2.63 (−10.32 to 5.04) 0.49
LDL (mg/dL) 135.42 ±2.76 127.64 ±2.78 −7.77 (−12.43 to −3.12) 0.001
SBP (mmHg) 131.02 ±0.77 131.00 ±0.81 −0.77 (−0.66 to 0.60) 0.93
DBP (mmHg) 78.57 ±0.51 78.25 ±0.55 −0.81 (−0.80 to 0.16) 0.19
Biochemical markers
25(OH)D (ng/mL) 20.73 ±0.36 32.14 ±0.59 11.41(10.27 to 12.54) <0.001
Calcium (mg/dL) 9.25 ±0.03 9.34 ±0.04 0.09 (0.006 to 0.18) 0.03
Phosphorus (mg/dL) 3.62 ±0.03 3.69 ±0.03 0.06 (−0.01 to 0.13) 0.10
PTH (pg/mL) 53.20 ±1.72 46.63 ±2.21 −6.57 (−11.08 to −2.05) 0.005
hs-CRP (mg/dL) 1.97 ±0.20 2.61 ±0.34 0.64 (−0.06 to 1.35) 0.07
IL-6 (pg/mL) 20.59 ±4.52 22.37 ±2.32 1.78 (−5.75 to 9.31) 0.64
Leptin (ng/mL) 25.93 ±1.57 24.68 ±1.45 −1.24 (−3.89 to 1.39) 0.35
Protein carbonyls (nmol/mg) 0.79 ±0.04 0.70 ±0.03 −0.08 (−0.16 to −0.003) 0.04
Abbreviations: F: female, M: male, BMI: body mass index, ASM: appendicular skeletal muscle mass, ASMI:
appendicular skeletal muscle mass index, SMI: skeletal muscle index, VAS: visual analogue scale, WOMAC: Western
Ontario and McMaster Universities Osteoarthritis Index, SF-12: 12-Item short form health survey, PCS: physical
health composite scores, MCS: mental health composite scores, PAQ-EJ: physical activity questionnaire for elderly
Japanese in Thai version, MET: metabolic equivalent of task, FBG: fasting blood glucose, HOMA-IR: homeostatic
model assessment of insulin resistance, HDL-cholesterol: high-density lipoprotein cholesterol, LDL-cholesterol:
low-density lipoprotein cholesterol, SBP: systolic blood pressure and DBP: diastolic blood pressure, 25(OH)D,
25-hydroxyvitamin D and PTH: parathyroid hormone.
Nutrients 2017,9, 799 6 of 13
3.2. Effects on Metabolic Risk Factors
HDL cholesterol levels increased after treatment, but the change was not statistically significant.
LDL cholesterol levels significantly decreased after vitamin D
2
supplementation (p= 0.001). FBG,
Insulin, HOMA-IR, and blood pressure did not change between baseline and six months, as shown in
Table 1.
3.3. Effects on Vitamin D and PTH Status
At baseline, the mean serum 25(OH)D level in knee OA patients was 20.73
±
0.36 ng/mL.
Seventy-two participants (41.10%) had vitamin D deficiency, and 103 patients (58.90%) had vitami D
insufficiency. After 40,000 IU of vitamin D
2
supplementation per week for six months, there was
a statistically significant increase in mean serum 25(OH)D level to 32.14
±
0.59 ng/mL (
p< 0.001
)
(Table 1). One hundred (57.10%) knee OA participants that previously had either vitamin D
insufficiency or deficiency at baseline achieved serum 25(OH)D concentration above 30 ng/mL (28 with
baseline deficiency and 72 with baseline insufficiency). After supplementation and at the six-month
time point, 70 knee OA participants (40.00%) had vitamin D insufficiency, and only five patients (2.90%)
had vitamin D deficiency (Figure 2). During treatment, levels of serum calcium increased significantly
(p< 0.05), three OA patients (1.71%) developed hypercalcemia (Ca > 10.5 mg/dL) and PTH decreased
significantly (p< 0.05) after vitamin D2supplementation.
Nutrients 2017, 9, 799 6 of 13
short form health survey, PCS: physical health composite scores, MCS: mental health composite
scores, PAQ-EJ: physical activity questionnaire for elderly Japanese in Thai version, MET: metabolic
equivalent of task, FBG: fasting blood glucose, HOMA-IR: homeostatic model assessment of insulin
resistance, HDL-cholesterol: high-density lipoprotein cholesterol, LDL-cholesterol: low-density
lipoprotein cholesterol, SBP: systolic blood pressure and DBP: diastolic blood pressure, 25(OH)D, 25-
hydroxyvitamin D and PTH: parathyroid hormone.
3.2. Effects on Metabolic Risk Factors
HDL cholesterol levels increased after treatment, but the change was not statistically significant.
LDL cholesterol levels significantly decreased after vitamin D2 supplementation (p = 0.001). FBG,
Insulin, HOMA-IR, and blood pressure did not change between baseline and six months, as shown
in Table 1.
3.3. Effects on Vitamin D and PTH Status
At baseline, the mean serum 25(OH)D level in knee OA patients was 20.73 ± 0.36 ng/mL.
Seventy-two participants (41.10%) had vitamin D deficiency, and 103 patients (58.90%) had vitamin
D insufficiency. After 40,000 IU of vitamin D2 supplementation per week for six months, there was a
statistically significant increase in mean serum 25(OH)D level to 32.14 ± 0.59 ng/mL (p < 0.001) (Table
1). One hundred (57.10%) knee OA participants that previously had either vitamin D insufficiency or
deficiency at baseline achieved serum 25(OH)D concentration above 30 ng/mL (28 with baseline
deficiency and 72 with baseline insufficiency). After supplementation and at the six-month time
point, 70 knee OA participants (40.00%) had vitamin D insufficiency, and only five patients (2.90%)
had vitamin D deficiency (Figure 2). During treatment, levels of serum calcium increased significantly
(p < 0.05), three OA patients (1.71%) developed hypercalcemia (Ca > 10.5 mg/dL) and PTH decreased
significantly (p < 0.05) after vitamin D2 supplementation.
Figure 2. Vitamin D status in knee OA patients at baseline and after vitamin D2 supplementation.
3.4. Effects on Inflammation, Adipokine and Oxidative Stress
Levels of hs-CRP, IL-6 and leptin were not different (p > 0.05), but protein carbonyls
concentration was significantly decreased between baseline and after vitamin D2 supplementation (p
= 0.04).
Figure 2. Vitamin D status in knee OA patients at baseline and after vitamin D2supplementation.
3.4. Effects on Inflammation, Adipokine and Oxidative Stress
Levels of hs-CRP, IL-6 and leptin were not different (p> 0.05), but protein carbonyls concentration
was significantly decreased between baseline and after vitamin D2supplementation (p= 0.04).
3.5. Effects on Muscle Strength and Physical Performance
Dominant grip strength (p= 0.01) and overall physical performance, such as gait speed (p< 0.001),
TUGT (p< 0.001), STS (p< 0.001), and 6MWT (p< 0.001), significantly improved after vitamin D
2
supplementation, but there were no significant difference observed for non-dominant grip strength
and knee extension force between baseline and post-treatment (p> 0.05) are presented in Table 2.
Nutrients 2017,9, 799 7 of 13
Table 2.
Muscle strength and physical performance at baseline, three months and six months after
vitamin D2supplementation.
Vitamin D2Supplementation (n= 175)
p-Value
Mean ±SEM
Baseline 3 Months 6 Months
Grip strength (kg)
Dominant (kg) 22.40 ±0.41 22.66 ±0.39 23.05 ±0.41 0.01
Non-dominant (kg) 20.26 ±0.40 20.09 ±0.38 20.45 ±0.40 0.13
Knee extension force:
Symptomatic leg (N) 356.01 ±5.95 354.84 ±5.32 358.61 ±5.38 0.31
Non-symptomatic leg (N) 378.22 ±5.84 378.00 ±5.65 379.90 ±5.79 0.45
Physical performances
Gait speed (m/s) 0.96 ±0.02 1.10 ±0.02 1.14 ±0.02 <0.001
TUGT (s) 9.81 ±0.19 8.81 ±0.20 8.65 ±0.17 <0.001
STS (s) 14.87 ±0.37 13.86 ±0.35 13.28 ±0.39 <0.001
6MWT (m) 371.22 ±5.95 400.05 ±6.32 421.20 ±5.83 <0.001
3.6. Association of 25(OH)D, Biochemical Markers and Body Composition
We found a negative correlation between 25(OH)D and IL-6 at baseline (r=
−
0.32, p< 0.001).
After vitamin D
2
supplementation, our results showed that 25(OH)D level was negatively correlated
with leptin (r=
−
0.20, p= 0.007), BMI (r=
−
0.24, p= 0.002) and fat mass (r=
−
0.20, p= 0.008) are
shown in Figure 3. Correlations between 25(OH)D level, muscle strength, and physical performance
were not significantly different between baseline and after treatment (p> 0.05).
Nutrients 2017, 9, 799 7 of 13
3.5. Effects on Muscle Strength and Physical Performance
Dominant grip strength (p = 0.01) and overall physical performance, such as gait speed (p <
0.001), TUGT (p < 0.001), STS (p < 0.001), and 6MWT (p < 0.001), significantly improved after vitamin
D2 supplementation, but there were no significant difference observed for non-dominant grip
strength and knee extension force between baseline and post-treatment (p > 0.05) are presented in
Table 2.
Table 2. Muscle strength and physical performance at baseline, three months and six months after
vitamin D2 supplementation.
Vitamin D2Supplementation (n= 175)
p-Value
Mean ± SEM
Baseline 3 Months 6 Months
Grip strength (kg)
Dominant (kg) 22.40 ± 0.41 22.66 ± 0.39 23.05 ± 0.41 0.01
Non-dominant (kg) 20.26 ± 0.40 20.09 ± 0.38 20.45 ± 0.40 0.13
Knee extension force:
Symptomatic leg (N) 356.01 ± 5.95 354.84 ± 5.32 358.61 ± 5.38 0.31
Non-symptomatic leg (N) 378.22 ± 5.84 378.00 ± 5.65 379.90 ± 5.79 0.45
Physical performances
Gait speed (m/s) 0.96 ± 0.02 1.10 ± 0.02 1.14 ± 0.02 <0.001
TUGT (s) 9.81 ± 0.19 8.81 ± 0.20 8.65 ± 0.17 <0.001
STS (s) 14.87 ± 0.37 13.86 ± 0.35 13.28 ± 0.39 <0.001
6MWT (m) 371.22 ± 5.95 400.05 ± 6.32 421.20 ± 5.83 <0.001
3.6. Association of 25(OH)D, Biochemical Markers and Body Composition
We found a negative correlation between 25(OH)D and IL-6 at baseline (r = −0.32, p < 0.001).
After vitamin D2 supplementation, our results showed that 25(OH)D level was negatively correlated
with leptin (r = −0.20, p = 0.007), BMI (r = −0.24, p = 0.002) and fat mass (r = −0.20, p = 0.008) are shown
in Figure 3. Correlations between 25(OH)D level, muscle strength, and physical performance were
not significantly different between baseline and after treatment (p > 0.05).
Figure 3. A negative association between 25(OH)D levels and biomarkers (a) IL-6 levels at baseline;
(b) leptin levels after vitamin D supplementation. The association between of vitamin D levels and
body composition after vitamin D supplementation; (c) BMI and (d) fat mass were negatively
associated with 25(OH)D levels.
Figure 3.
A negative association between 25(OH)D levels and biomarkers (
a
) IL-6 levels at baseline;
(
b
) leptin levels after vitamin D supplementation. The association between of vitamin D levels and body
composition after vitamin D supplementation; (
c
) BMI and (
d
) fat mass were negatively associated
with 25(OH)D levels.
Nutrients 2017,9, 799 8 of 13
4. Discussion
The objective of this study was to determine whether vitamin D supplementation could improve
muscle strength and physical performance in knee OA patients with low vitamin D status. The results
showed that knee OA with vitamin D
2
supplementation improved grip strength and physical
performance, but did not improve knee extension force. We also found that vitamin D supplementation
reduced oxidative protein damage, reduced pain, and improved quality of life.
Six months after supplementation of 40,000 IU of vitamin D
2
per week, 57% of patients
achieved vitamin D sufficiency, whereas 40% and 3% had vitamin D insufficiency and deficiency,
respectively. Generally, the source of vitamin D supplementation from diet and dietary supplements
are ergocalciferol (vitamin D
2
) and cholecalciferol (vitamin D
3
), which are inactive forms of vitamin D.
Vitamin D
2
are found plant and yeast irradiation, whereas the sources of vitamin D
3
are oily fish
and meat [
24
]. Similarly, two types of vitamin D supplementation are available for over-the-counter
purchase. In Thailand, ergocalciferol is used to treat vitamin D deficiency as the first-line therapeutic
drug. However, some evidence suggests that vitamin D
2
should not be regarded as equivalent to
vitamin D
3
for maintaining the concentration of 25(OH)D [
25
]. Serum 25(OH)D
2
has a lower affinity for
vitamin D-binding protein (DBP), and the serum half-life of 25(OH)D
2
is shorter than 25(OH)D
3
[
26
].
There is possibly a higher affinity of hepatic 25-hydroxylase for vitamin D
3
than for vitamin D
2
[
27
].
The results of our study have demonstrated that 40,000 IU of vitamin D
2
per week was able to enhance
25(OH)D levels to achieve vitamin D sufficiency in only 57% of participants. In fact, other factors may
influence the increment of vitamin D levels, such as dietary vitamin D intake and exposure to the
sunlight, which were not included in this study. According to the experimental design in this study,
only one group of the population was deployed to study the effect of vitamin D supplementation
between before and after supplementation. We believed that they would have been exposed to
an equivalent amount of sunlight and consumed vitamin D-containing food in similar amounts before
and after supplementation due to their daily behaviors.
Vitamin D
2
supplementation also affected calcium and PTH levels. We found that serum Ca levels
increased and PTH levels decreased significantly after supplementation. Only 1.71% (n= 3) of cases had
mild hypercalcemia after vitamin D
2
supplementation. Pietras et al. reported no incidents of vitamin D
toxicity and normal levels of serum calcium in patients who were treated with 50,000 IU of vitamin D
2
every other week for up to six years [
28
]. Del Valle et al. studied a high-dose ergocalciferol 72,000 IU/week
for 12 weeks and maintenance therapy 24,000 IU/week during 36 weeks in hemodialysis patients. They
found that only 1.8% had hypercalcemia [
29
]. However, blood calcium levels are not a good reflection
of calcium status, whereas urinary calcium excretion determines the risks of vitamin D treatment for
excessive calcium absorption. Consequently, the results revealed that PTH levels significantly decreased
after treatment. The previous study reported that low vitamin D status was associated with elevated
bone turnover by increasing PTH levels [
30
]. Moreover, high levels of PTH are related with the risk of
fall, fracture, and poorer outcomes in terms of frailty [
31
]. PTH action stimulates the transformation
of pro-osteoclasts into mature osteoclasts, which leads to increasing bone turnover [
32
]. Consequently,
optimal vitamin D levels may help to reduce the risk of fall, fracture, and osteoporosis.
Body composition, including weight, percentage of fat, fat mass and visceral fat rating, all decreased
significantly after vitamin D
2
supplementation compared with their baseline values, but skeletal muscle
mass did not change. Our results showed that the participants lost weight, which might be due to
change in their lifestyles, and had significantly improved physical function according to increasing
physical health composite scores (PCS) of SF-12, while physical activity assessments from PAQ-EJ did
not differ. Moreover, we also observed weak negative association between both 25(OH)D and BMI
and 25(OH)D and fat mass after vitamin D supplementation. Consistent with our result, Lagari et al.
reported that higher fat mass was associated with lower vitamin D status [
33
]. Therefore, patients with
a higher BMI or obesity may experience slower increases in serum vitamin D level than people with
normal or thin body composition. This suggests that higher doses of vitamin D supplementation and
longer treatment times may be needed in knee OA patients with higher BMI or obesity.
Nutrients 2017,9, 799 9 of 13
Self-reported pain and health-related quality of life showed improvement after vitamin D
supplementation according to results obtained from VAS and the PCS of SF-12 questionnaires.
However, WOMAS score is not relevant. The previous study reported that WOMAC and VAS
decreased significantly after vitamin D supplementation [
5
]. In contrast, other studies reported that
vitamin D supplementation did not reduce knee pain, cartilage volume loss, or improve physical
function [
14
,
34
]. Actually, VAS assessed severity of pain from the patient’s perspective at the moment
of assessment, and the pain VAS is a single-item scale. The WOMAC score used in the evaluation of
knee OA consists of three subscales such as pain, stiffness and physical function (the questions cover
everyday activities). Therefore, the effect of vitamin D supplementation on VAS may not be a good
reflection of pain during daily activities.
The effect of vitamin D supplementation on metabolic risk factors presented a significant reduction
in LDL-cholesterol. The participants had lost weight, decreased fat percentages, and lower fat mass
and visceral fat ratings, which may have result in the reduced LDL-cholesterol levels in this study.
The previous studies have shown a significant reduction in LDL-cholesterol levels after vitamin D
supplementation [
35
,
36
]. The effects of vitamin D increase level of intestinal calcium intake, while
calcium may reduce fatty acid absorption due to the formation of insoluble calcium–fatty complexes in
the gut. Therefore, serum levels of LDL-cholesterol would be decreased by the reduced absorption
of saturated fatty acids [
37
]. However, vitamin D supplementation did not improve lipid profiles in
obese individuals [38,39].
In regards to the relationship between vitamin D, inflammation, and adipokine, the results
demonstrated a weak negative association of 25(OH)D with IL-6 and leptin. These results were consistent
with a previous report that vitamin D deficiency was associated with more pro-inflammatory cytokines
as compared with insufficiency or sufficiency status in elderly adults [
40
]. Moreover, the previous studies
reported a negative association between serum 25(OH)D and leptin concentrations [41,42].
In addition, our data showed that vitamin D supplementation reduced oxidative protein
damage by decreasing levels of protein carbonyl. Protein carbonyl was used as the biomarker of
oxidative damage, since it leads to cellular dysfunction and a decline in muscle function [
43
,
44
].
It is the mechanism involved in the direct oxidation of amino acids such as lysine, arginine,
histidine, proline, glutamic acid, and threonine, or by the binding of aldehydes produced from
lipid peroxidation [
23
]. Carbonyl stress can modify protein function and cause DNA damage through
stimulating pro-inflammatory signaling (nuclear factor-kB: NF-kB & p38), tissue remodeling, muscle
dysfunction, [
45
] and the pathogenesis of sarcopenia [
46
]. Vitamin D may be regarded as an antioxidant
in which 1,25-dihydroxyvitamin D (1,25(OH)
2
D) binding to the vitamin D receptor (VDR) and the
retinoid X receptor (RXR) interact with various nuclear co-activators that regulate gene transcription.
It may reduce reactive oxygen species formation by the suppression of the gene expression of
NADPH oxidase, and induce the expression of antioxidant genes [
47
]. Moreover, 1,25(OH)
2
D has
been demonstrated to suppress the production of pro-inflammatory cytokines, such as IL-6 and tumor
necrosis factor-
α
(TNF-
α
), as well as reduce the expression of NF-kB and p38 [
48
]. These findings
suggest that high levels of vitamin D after supplementation may reduce the amounts of reactive oxygen
species produced by damaging proteins.
Regarding muscle strength and physical performance, we found that knee OA patients
significantly improved grip strength and physical performance, but did not improve knee extension
force. In this aspect, our results are consistent with the findings of several previous studies. Zhu et al.
reported that hip muscle strength and TUGT improved significantly after 1000 IU/day vitamin D
2
supplementation for one year in older women with vitamin D insufficiency [
49
]. Lagari et al. reported
that vitamin D supplementation might be most beneficial in older populations with poor physical
function [
33
]. Sato et al. found that the mean of type II muscle fiber diameter and percentage
of type II fibers increased significantly after 1000 IU/day vitamin D
2
treatment over two years in
elderly patients with post-stroke hemiplegia [
50
]. Ceglia et al. reported that intramyonuclear VDR
concentration increased 30% and total (type I and II) muscle fiber size increased 10% after vitamin D
Nutrients 2017,9, 799 10 of 13
supplementation in mobility-limited elderly women [
51
]. However, some studies have reported
that vitamin D supplementation did not improve muscle strength or physical function. Kenny et al.
found that vitamin D supplementation did not improve muscle strength or physical performance in
a group of healthy community-dwelling older men [
11
]. These conflicting findings may be attributed
to differences in populations, disease advancement, or measurements applied, or to incomplete control
of confounding variables. Nonetheless, conclusions should be drawn with caution on whether the
characteristics of studied participants or the dose of vitamin D used are of significance, as these studies
were heterogeneous with regards to most aspects. Various outcome measures have been documented
by different investigators and even in the case of measurements of similar characteristics, different
methods have been applied, making it difficult to compare studies directly.
A strength of this study is the finding that a high dose and a long-term intervention of vitamin D
2
supplementation was effective in raising 25(OH)D concentrations. It is possible that achieved serum
25(OH)D levels may improve muscle function by increasing muscle strength and physical performance
in knee OA patients. Higher serum 25(OH)D concentrations may be essential in skeletal muscle,
particularly for the elderly with limited mobility [
33
,
50
,
51
]. On the other hand, increasing 25(OH)D
levels in healthy populations do not relate to any improvement of muscle function [
11
]. Therefore,
patients with impaired mobility may be more sensitive to the improvement in physical functioning by
vitamin D supplementation. Previous studies indicated that vitamin D supplementation in the elderly
with vitamin D insufficiency reduced an atrophy of type II muscle fiber [
50
] and increased the size of type
I and II muscle fiber, as well as VDR concentration [
51
]. Actually, knee OA patients with poor muscle
function and vitamin D deficiency may be the most likely to benefit from vitamin D supplementation.
This study has several mentionable limitations. First, the controlled before–after design of this
study did not include a control group. The lack of randomization, and our decision not to evaluate the
sensitivity of drug effect, potentially weaken our findings relative to the therapeutic effect of vitamin D
supplementation. Second, the sample size was small and the proportion of men was low, both of
which prevented us from establishing the clinical relevance, particularly regarding changes in muscle
strength. Third, we assayed markers of oxidative damage using plasma protein carbonyls that were
not directly measured in skeletal muscle. Finally, 8.37% of patients were lost to follow-up. While this
rate is higher than can be considered ideal, the loss to follow-up rate in the present study was lower
than loss to follow-up rates reported from other studies.
5. Conclusions
In conclusion, our results suggest that 40,000 IU of vitamin D
2
supplementation reduced oxidative
protein damage, improved quality of life, and improved grip strength and physical performance.
It remains unclear whether vitamin D supplementation relates to musculoskeletal pain or not. Accordingly,
vitamin D treatment decreases current pain using VAS, but does not reduce pain during physical activity,
as determined by WOMAC score. Nevertheless, vitamin D supplementation is a safe and inexpensive
way to improve muscle strength and physical function in this population. Based on these findings, we
recommend vitamin D supplementation in knee OA patients that have poor physical function.
Acknowledgments:
This study was supported by research grants from the 90th Anniversary Chulalongkorn
University Fund, the University of Phayao Fund, and National Research University Project, Office of Higher
Education Commission through Aging Society Cluster (NRU59-056-AS), Chulalongkorn University. The authors
wish to thank the nurses and staff of the Department of Orthopaedics, King Chulalongkorn Memorial Hospital
for their support of this study and Borwarnluck Thongtha, Surasit Suwannasin, Patcharawalai Wongsiri and
Nungruthai Nilsri for excellent technical assistance. We also thank Kevin P. Jones for proof-reading the manuscript.
Author Contributions:
Pacharee Manoy, Pongsak Yuktanandana, Aree Tanavalee, Wilai Anomasiri and
Sittisak Honsawek conceived and designed the experiments; Pacharee Manoy performed the experiments;
Pacharee Manoy, Pongsak Yuktanandana, Aree Tanavalee, Wilai Anomasiri, Srihatach Ngarmukos,
Thanathep Tanpowpong and Sittisak Honsawek analyzed the data; Sittisak Honsawek contributed
reagents/materials/analysis tools; Pacharee Manoy, Wilai Anomasiri and Sittisak Honsawek wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2017,9, 799 11 of 13
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