Annals of Internal Medicine Original Research

High Doses of Vitamin D to Reduce Exacerbations in Chronic

Obstructive Pulmonary Disease
A Randomized Trial
An Lehouck, PhD; Chantal Mathieu, MD, PhD; Claudia Carremans, MS; Femke Baeke, PhD; Jan Verhaegen, MD, PhD;
Johan Van Eldere, MD, PhD; Brigitte Decallonne, MD, PhD; Roger Bouillon, MD, PhD; Marc Decramer, MD, PhD; and
Wim Janssens, MD, PhD

Background: Low serum 25-hydroxyvitamin D (25-[OH]D) levels Results: Mean serum 25-(OH)D levels increased significantly in the
have been associated with lower FEV1, impaired immunologic con- vitamin D group compared with the placebo group (mean
trol, and increased airway inflammation. Because many patients between-group difference, 30 ng/mL [95% CI, 27 to 33 ng/mL];
with chronic obstructive pulmonary disease (COPD) have vitamin D P ⬍ 0.001). The median time to first exacerbation did not signifi-
deficiency, effects of vitamin D supplementation may extend be- cantly differ between the groups (hazard ratio, 1.1 [CI, 0.82 to
yond preventing osteoporosis. 1.56]; P ⫽ 0.41), nor did exacerbation rates, FEV1, hospitalization,
quality of life, and death. However, a post hoc analysis in 30
Objective: To explore whether supplementation with high doses of participants with severe vitamin D deficiency (serum 25-[OH]D
vitamin D could reduce the incidence of COPD exacerbations. levels ⬍10 ng/mL) at baseline showed a significant reduction in
Design: Randomized, single-center, double-blind, placebo-controlled exacerbations in the vitamin D group (rate ratio, 0.57 [CI, 0.33 to
trial. (ClinicalTrials.gov registration number: NCT00666367) 0.98]; P ⫽ 0.042).

Setting: University Hospitals Leuven, Leuven, Belgium. Limitation: This was a single-center study with a small sample size.

Patients: 182 patients with moderate to very severe COPD and a Conclusion: High-dose vitamin D supplementation in a sample of
history of recent exacerbations. patients with COPD did not reduce the incidence of exacerbations.
In participants with severe vitamin D deficiency at baseline, supple-
Intervention: 100 000 IU of vitamin D supplementation or placebo mentation may reduce exacerbations.
every 4 weeks for 1 year.
Primary Funding Source: Applied Biomedical Research Program,
Measurements: The primary outcome was time to first exacerba- Agency for Innovation by Science and Technology (IWT-TBM).
tion. Secondary outcomes were exacerbation rate, time to first
hospitalization, time to second exacerbation, FEV1, quality of life, Ann Intern Med. 2012;156:105-114. www.annals.org
and death. For author affiliations, see end of text.

C hronic obstructive pulmonary disease (COPD) is

characterized by an abnormal inflammatory response
of the airways to the inhalation of noxious particles or
reported disappointing results. A reason for these negative
results may be found in insufficient vitamin D supplemen-
tation, because no clear evidence nor consensus exists on
gases, such as cigarette smoke. With disease progression, what minimum serum levels are needed to achieve these
marked by a decline in FEV1, patients develop systemic “extraskeletal” effects. On the basis of observational data,
consequences and become prone to infectious exacerba- experts have suggested that in contrast to obtaining bene-
tions (1). Of note, low serum 25-hydroxyvitamin D (25- ficial effects on the bone, higher serum levels (30 to 50
[OH]D) levels, reflecting vitamin D status, are associated ng/mL) are needed, requiring more aggressive supplemen-
with impaired FEV1 (2), and we demonstrated that vita- tation regimens (15–17). One recent intervention study in
min D deficiency (defined as serum 25-[OH]D levels ⬍20 tuberculosis corroborates with this idea (18). Overall, an
ng/mL by the Institute of Medicine [3]), is present in 60%
to 75% of patients with severe COPD (4). Whether such
deficiency is only the consequence of COPD or may caus- See also:
ally contribute to the pathogenesis of COPD is unclear.
Print
Traditionally, vitamin D is associated with bone
Editors’ Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
health (5), but large epidemiologic studies have associated
Editorial comment. . . . . . . . . . . . . . . . . . . . . . . . . . 156
low serum 25-(OH)D levels with autoimmune diseases;
Summary for Patients. . . . . . . . . . . . . . . . . . . . . . . I-26
cancer; cardiovascular diseases; and infections, including
respiratory tract infections and tuberculosis (6 –10). How- Web-Only
ever, few randomized, controlled trials have examined the Appendix
effects of vitamin D supplementation on these important Appendix Tables
health outcomes. Moreover, recent studies of vitamin D Appendix Figures
supplementation in patients with multiple sclerosis (11), Conversion of graphics into slides
diabetes (12), influenza (13), and tuberculosis (14) have
© 2012 American College of Physicians 105

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 Original Research High Doses of Vitamin D to Reduce COPD Exacerbations

Context
(21). All participants provided written informed consent.
The study was approved by the local ethics review commit-
The association between low serum vitamin D levels and
tee of the University Hospitals Leuven and was registered
more severe chronic obstructive pulmonary disease
with ClinicalTrials.gov (NCT00666367).
(COPD) suggests that vitamin D supplementation might
be beneficial for COPD treatment. Randomization and Masking
Randomization was performed in 2 strata: one group
Contribution
of vitamin D–naive participants and one group of partici-
In this randomized trial, supplementation with vitamin D pants receiving low-dose vitamin D supplements (400 to
did not reduce the number of acute exacerbations of 880 IU/d) for osteoporosis at baseline. We randomly as-
COPD nor improve lung function, compared with placebo.
signed participants in blocks of 20 to overcome seasonal
A post hoc analysis suggested possible benefit in patients
influences on baseline characteristics. In each consecutive
with the lowest baseline vitamin D levels.
block, participants were allocated in a 1:1 ratio to receive a
Implication monthly oral dose of 100 000 IU of vitamin D (4 mL of
Vitamin D supplementation does not seem to be beneficial D-Cure [manufactured and provided by Laboratoires
for COPD, although further study may be warranted to SMB, Brussels, Belgium]) or placebo (4 mL of arachidis
assess whether it might help certain patient groups. oleum) in addition to their regular treatment. Pharmacists
of the University Hospitals Leuven, who were independent
—The Editors from the clinical study team, randomly assigned partici-
pants by using a computer-generated randomization list
and prepared the study medication. Vitamin D and pla-
extensive expert analysis of the potential effects of vita- cebo were prepared in oral syringe dispensers, were identi-
min D supplementation on the health outcomes of cal in appearance and taste, and were numbered according
North American participants concluded that evidence to the randomization schedule. After the last participant
for extraskeletal benefits of vitamin D therapy is insuf- completed the trial, masking continued until all data were
ficient and that only new randomized, controlled trials entered in a database, which was verified and locked before
can define such effects (19). unblinding in July 2010.
Particularly for COPD, the vitamin D pathway is an Procedures
attractive target for intervention studies because vitamin D
Patients were screened during hospitalization for an
deficiency may enhance chronic airway and systemic in-
exacerbation or before referral for respiratory rehabilita-
flammation, reduce bacterial clearance, and increase the
tion. Randomization occurred 5 to 6 weeks after screening
risk for infectious exacerbations at the same time (20).
if the participant had convalesced. If not, randomization
Therefore, we aimed to explore the effect of adequate vita-
was postponed until steroid, antibiotic, or combination
min D supplementation on exacerbations in patients with
treatment was completed and spirometric values were
moderate to very severe COPD. We report the efficacy and
similar to preexacerbation values. Baseline characteristics
safety of long-term, high-dose vitamin D supplementation
included the Body-Mass Index, Airflow Obstruction, Dys-
in patients with COPD prone to exacerbations.
pnea, and Exercise Capacity (BODE) Index, a multi-
dimensional, COPD-specific, 10-point scale in which
METHODS higher scores indicate a higher risk for death (22), and the
Study Design and Participants Charlson comorbidity index, a non–COPD-specific but
Our study was a single-center, double-blind, random- validated comorbidity index (23). After randomization,
ized, placebo-controlled intervention trial. Patients were follow-up visits occurred every 4 months (at 4, 8, and 12
screened for eligibility at the University Hospitals Leuven, months). The primary end point was the time to first ex-
Leuven, Belgium, over a 1.5-year recruitment period in acerbation. Secondary end points were exacerbation rate;
2008 and 2009. Eligible patients were current or former time to first hospitalization; time to second exacerbation;
smokers, were older than 50 years, had a diagnosis of FEV1 (24); quality of life, as measured with the Chronic
COPD according to the Global Initiative for Chronic Ob- Respiratory Questionnaire (CRQ) (scores for dyspnea,
structive Lung Disease (GOLD) definition (postbroncho- emotion, fatigue, and mastery) (25); and death. In addition
dilator FEV1–FVC ratio ⬍0.7), and had an FEV1 less than to these clinical end points, bacterial presence in morning
80% predicted. Patients were excluded if they had a history sputa, plasma cathelicidin levels, serum 25-(OH)D levels,
of hypercalcemia, sarcoidosis, or active cancer. Treatment and blood monocyte capacities for phagocytosis were de-
with vitamin D supplements for newly discovered symp- termined in a blinded manner (Appendix, available at
tomatic osteoporosis and long-term azithromycin treat- www.annals.org).
ment, with antibacterial and anti-inflammatory functions, The clinical study team remained blinded to these re-
were additional exclusion criteria, because they could inter- sults until the database was locked. We defined COPD
fere with 25-(OH)D dosages and exacerbation analyses exacerbations as sustained worsening of respiratory symp-
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 High Doses of Vitamin D to Reduce COPD Exacerbations Original Research

toms during 48 hours and requiring oral corticosteroid, compared by using Kaplan–Meier curves and log-rank
antibiotic, or combination treatment that was initiated by tests. We calculated the mean number of exacerbations per
a physician. Respiratory symptoms included at least 1 of patient-year by dividing the total number of exacerbations
the Anthonisen criteria (26) (increased dyspnea, sputum by the total years of follow-up in the ITT population.
volume, or sputum purulence) with or without minor Exacerbation rate in the ITT population was analyzed with
symptoms, such as cough, fever, common cold, wheezing, a generalized linear model for a Poisson distribution, cor-
or sore throat. Time to first exacerbation was assessed by recting for duration of treatment exposure and overdisper-
quantifying the days between randomization and the first sion. Because age, FEV1, GOLD stage, and smoking status
exacerbation. To obtain data on exacerbations, we asked did not have a statistically significant influence on our
participants to complete diaries every 2 weeks that detailed model, we did not include these covariates in the final
respiratory tract symptoms, visits to health care providers, analysis (29). In a post hoc analysis for exacerbation rate, a
hospitalizations, and changes in medication. At each visit, similar generalized linear model for a Poisson distribution
diaries were reviewed in the participant’s presence and the
was applied in the ITT population but with correction for
general practitioner was contacted in case of doubt, missing
baseline serum 25-(OH)D levels and for the interaction of
data, or suspicion of self-medication.
these levels with treatment. All further subgroup analyses
Spirometry was repeated at each visit by using stan-
dard equipment (CareFusion, Vilvoorde, Belgium) and (time to first exacerbation, exacerbation rate, and serum
was performed according to American Thoracic Society/ 25-[OH]D levels) in participants receiving low-dose
European Respiratory Society guidelines (27). To monitor supplements, vitamin D–naive participants at baseline,
safety, we collected blood samples every 4 months to mea- or participants with severe vitamin D deficiency (de-
sure serum calcium and phosphate levels. After a protocol fined as having serum 25-[OH]D levels ⬍10 ng/mL)
amendment halfway through the trial, we collected urine were post hoc.
samples to better document the safety profile of the study Serial FEV1, CRQ scores, plasma cathelicidin levels,
drug. From that point on, all participants (n ⫽ 79) col- serum 25-(OH)D levels, and bacterial presence in morning
lected 24-hour urine the day before the last study visit to sputa over 1 year were predefined secondary end points.
measure urinary calcium level, the calcium– creatinine ra- They were compared in a linear mixed-model analysis,
tio, and the glomerular filtration rate. with visit number as the repeated measure and the respec-
tive marker as the dependent variable, in the ITT popula-
Statistical Analysis tion. Two-way analysis-of-variance P values of treatment-
The study was designed to demonstrate a minimum by-visit interaction are reported. Posttests to compare
delay of 25% in the time to first exacerbation when com- differences per visit were performed by using t test statistics
paring the vitamin D group with the placebo group. As we with unadjusted P values. Chi-square statistics were used to
enrolled patients who were recently treated for an exacer- compare proportions of participants with hypercalcemia
bation, we based our assumptions on the MOSAIC (Moxi- (during the study) and hypercalciuria (at the end of the
floxacin Compared to Standard Antibiotics for Acute Ex- study) between the groups at every visit. To compare the
acerbations in Chronic Bronchitis) trial (28), which best
monocyte capacity for phagocytosis between the groups at
resembled our study sample and showed a mean time to
the end of the study, we used t test statistics. Subgroup
next exacerbation of 130 days (SD, 70) after a first exacer-
analysis of monocyte capacity for phagocytosis and plasma
bation. Based on t test statistics, a sample size of 57 par-
ticipants in each group was needed to demonstrate a 25% cathelicidin levels in participants with severe vitamin D
delay in time to first exacerbation, with 80% power at 5% deficiency at baseline were post hoc analyses. Effect sizes
significance. Because approximately 20% of our partici- between groups of primary or secondary outcomes are
pants were receiving low-dose vitamin D supplements for given with P values and 95% CIs. P values less than 0.05
osteoporosis prevention (which was not an exclusion crite- are considered statistically significant. Analyses were per-
rion), and given an estimated maximum rate of 15% of formed with SAS software, version 9.1 (SAS Institute,
participant withdrawals without follow-up, 180 partici- Cary, North Carolina), and GraphPad Prism 4.01 for
pants had to be randomly assigned to end up with at least Windows (GraphPad Software, La Jolla, California).
120 vitamin D–naive participants at inclusion. All analyses
for exacerbations, deaths, and serum 25-(OH)D levels used Role of the Funding Source
the intention-to-treat (ITT) population, defined as all ran- The Applied Biomedical Research Program, Agency
domly assigned participants who received at least 1 dose of for Innovation by Science and Technology (IWT-TBM),
study medication. All available data were used for the ITT provided funding for the study, and Laboratoires SMB
analysis. On-treatment analyses for these variables are re- provided the study medication. These sources were not
ported in the Appendix. involved in the study design; collection, analysis, or inter-
Time to first or second exacerbation and time to first pretation of data; or in prepartion or submission of the
hospitalization in both groups in the ITT population were manuscript for publication.
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 Original Research High Doses of Vitamin D to Reduce COPD Exacerbations

Figure 1. Study flow diagram.

Patients were assessed for

eligibility (n = 419)

Withdrew during
screening or did not
meet entry criteria
(n = 237)

Randomly assigned
(n = 182)

Were assigned to Were assigned to

receive vitamin D receive placebo
(n = 91) (n = 91)

Deaths (n = 9) Deaths (n = 6)

Excluded (n = 10) Excluded (n = 7)

Withdrew consent: 3 Withdrew consent: 3
Did not adhere to Did not adhere to
protocol: 1 protocol: 1
Other reasons: 6 Other reasons: 3

Completed the study Completed the study

(n = 72) (n = 78)

Included in ITT Included in ITT

analysis analysis
(n = 91) (n = 91)

ITT ⫽ intention-to-treat.

RESULTS tion rate). We did not have follow-up data on exacerba-

Study Recruitment and Follow-up tions for the remaining 7 participants, but we still had
Figure 1 shows the study flow diagram. Of 419 access to their files to complete data on deaths. Overall, we
screenings, 340 patients were eligible for inclusion; 182 collected information on exacerbations and survival in 175
(54%) were randomly assigned. One hundred fifty (82%) (96%) and 182 (100%) participants, respectively.
participants completed the study, 15 (8%) died, and 17
(9%) were classified as withdrawals with no differential Baseline Characteristics
dropout between the 2 groups. Sixteen participants com- The Table shows the baseline characteristics of the
pleted follow-up but were removed from a prespecified participants. Study inclusion required that the participant
on-treatment analysis for serum 25-(OH)D levels because not be receiving antibiotic and oral steroid treatment for an
they had started vitamin D supplementation or long-term acute exacerbation. However, 14% of the participants were
azithromycin treatment during the study (Appendix Fig- considered to be steroid-dependent, and the stable pretrial
ure 1, available at www.annals.org). Among the 17 with- maintenance dose (4 mg of methylprednisolone) was contin-
drawals, medication was stopped, but the study team con- ued during the study. Eighty-two percent of the participants
tinued collecting data on exacerbations and deaths. From were receiving maximum inhalation therapy (inhaled cortico-
10 of the 17 participants, we were able to contact the steroid plus long-acting ␤-agonist plus long-acting anticholin-
general practitioner and collected information on deaths ergic drug therapy) before inclusion; this proportion did not
and exacerbations (time to first exacerbation and exacerba- change during or at the end of the study (84%). Forty (22%)
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 High Doses of Vitamin D to Reduce COPD Exacerbations Original Research

participants were receiving vitamin D supplements (800 the time to first exacerbation and annual rate of exacerba-
IU/d) for bone protection before randomization, but they tions or other outcomes did not differ in this subgroup
were equally distributed between the groups (20 persons (Figure 3, B, and Appendix Table 2). Data from the sub-
each). group of participants receiving low-dose supplements at
Exacerbations baseline are not reported.
A total of 468 exacerbations occurred: 229 in the vi-
tamin D group and 239 in the placebo group. Kaplan– Interaction Between Vitamin D Supplementation and
Meier survival analysis showed no significant difference in Baseline Serum 25-(OH)D Levels
the median time to first exacerbation between the vitamin Because a post hoc analysis on the rate of exacerba-
D and placebo groups (84 days [interquartile range {IQR}, tions in the ITT population demonstrated a significant
29 to 200 days] vs. 56 days [IQR, 21 to 200 days]) (hazard interaction in treatment by baseline serum 25-(OH)D level
ratio [HR], 1.1 [95% CI, 0.82 to 1.56]; P ⫽ 0.41) (Figure (P ⫽ 0.027), we performed a further subgroup analysis for
2, A). Median time to second exacerbation also did not participants with severe vitamin D deficiency at baseline
differ between the groups (204 days [IQR, 123 to 329 (serum 25-[OH]D levels ⬍10 ng/mL). Among these 30
days] vs. 201 days [IQR, 113 to 333 days]) (HR, 1.02 [CI, participants, 15 were randomly allocated to receive vi-
0.72 to 1.47]; P ⫽ 0.88) (Figure 2, B). The annual rate of tamin D supplementation, which resulted in a signifi-
exacerbations was 2.8 per patient-year in the vitamin D cant increase in serum 25-(OH)D levels (from 8 ng/mL
group and 2.9 in the placebo group, resulting in a nonsig- [SD, 2] to 50 ng/mL [SD, 15]; mean between-group
nificant rate ratio of 0.94 (CI, 0.76 to 1.16; P ⫽ 0.57). We difference, 38 ng/mL [CI, 33 to 44 ng/mL]; P ⬍
did not find a significant difference in the median time to 0.001). Although the time to first exacerbation did not
first hospitalization for an exacerbation (HR, 0.84 [CI, differ in this subgroup, the rate of exacerbations per
0.50 to 1.40]; P ⫽ 0.50) (Figure 2, C). A total of 152 patient-year decreased by 43% (rate ratio, 0.57 [CI,
exacerbations resulted in hospitalization: 79 in the vitamin 0.33 to 0.98]; P ⫽ 0.042) (Figure 3, C).
D group and 73 in the placebo group (rate ratio, 1.13 [CI,
0.70 to 1.82]; P ⫽ 0.62).
Death, FEV1, and Quality of Life
Table. Baseline Characteristics*
Fifteen deaths occurred within 1 year after randomiza-
tion: 11 of respiratory disease, 1 of lung cancer, 2 of car-
Characteristic Vitamin D Group Placebo Group
diac disease, and 1 of unknown cause. The proportions of (n ⴝ 91) (n ⴝ 91)
deaths from any cause were 10% in the vitamin D group Men, n (%) 72 (79) 73 (80)
and 7% in the placebo group (rate ratio, 1.5 [CI, 0.56 to Mean age (SD), y 68 (9) 68 (8)
4.04]; P ⫽ 0.42). Kaplan–Meier survival analysis showed Mean BMI (SD), kg/m2 25 (5) 24 (5)
Smoking status
no significant difference in survival between the groups Current smokers, n (%) 13 (14) 19 (21)
(HR, 0.69 [CI, 0.25 to 1.90]; P ⫽ 0.47) (Figure 2, D). Mean pack-years smoked (SD) 51 (23) 53 (32)
Linear mixed-model analysis showed no significant differ- Mean FEV1 (SD), L 1.22 (0.45) 1.17 (0.43)
Mean FEV1 (SD), % predicted 44 (16) 42 (14)
ences in CRQ dyspnea, emotional, mastery, and fatigue Mean FVC (SD), L 2.78 (0.78) 2.88 (0.83)
scores and in FEV1 between the group at any time during Mean FEV1–FVC ratio (SD), % 44 (12) 41 (11)
follow-up (Appendix Figure 2 and Appendix Table 1, Mean DLCO (SD), % predicted 46 (16) 49 (16)
GOLD stage, n (%)
available at www.annals.org). II 25 (28) 24 (26)
Efficacy of Supplementation III 43 (47) 48 (53)
IV 23 (25) 19 (21)
Serum 25-(OH)D levels in the ITT population were Mean 25-(OH)D level (SD), ng/mL 20 (12) 20 (11)
measured during 90% of the visits. At baseline, mean se- Mean Charlson index score (SD) 3.48 (2.54) 3.52 (2.44)
Mean BODE Index score (SD) 3.82 (2.28) 3.55 (2.03)
rum 25-(OH)D levels did not differ between the vitamin
Medication, n (%)
D group (20 ng/mL [SD, 12]) and the placebo group (20 LABA 2 (2) 4 (4)
ng/mL [SD, 11]) (Figure 3, A; and Appendix Table 2, LABA plus ICS 81 (89) 79 (87)
Long-acting anticholinergic drugs 81 (89) 84 (92)
available at www.annals.org). Vitamin D supplementation
Short-acting bronchodilators 64 (70) 72 (79)
resulted in a steep and significant increase in serum 25- Steroids† 11 (12) 15 (16)
(OH)D levels that remained stable during the study (52 Long-term oxygen therapy 11 (12) 10 (11)
Started with rehabilitation at first visit 25 (27) 25 (27)
ng/mL [SD, 16]) (mean between-group difference, 30
ng/mL [CI, 27 to 33 ng/mL]; P ⬍ 0.001) (Figure 3, A, 25-(OH)D ⫽ 25-hydroxyvitamin D; BMI ⫽ body mass index; BODE ⫽ Body-
and Appendix Table 2). A post hoc analysis in the sub- Mass Index, Airflow Obstruction, Dyspnea, and Exercise Capacity; DLCO ⫽ dif-
fusing capacity of lung for carbon monoxide; GOLD ⫽ Global Initiative for
group of vitamin D–naive participants (n ⫽ 142) demon- Chronic Obstructive Lung Disease; ICS ⫽ inhaled corticosteroid; LABA ⫽ long-
strated that mean serum 25-(OH)D levels at baseline were acting ␤-agonist.
* Baseline characteristics of vitamin D and placebo groups at randomization. Both
lower than those of the ITT population, but that the sub- groups were matched for all given variables.
group received equal, effective supplementation. However, † ⱕ4 mg of methylprednisolone.

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 Original Research High Doses of Vitamin D to Reduce COPD Exacerbations

Figure 2. Kaplan–Meier plots of time to first exacerbation (A), time to second exacerbation (B), time to first hospitalization (C),
and death from any cause (D) in the intention-to-treat population.

A 100 B 100

Vitamin D

Probability of Second
80 80
Probability of First

Placebo

Exacerbation, %
Exacerbation, %

60 60

40 40
Vitamin D
Placebo
20 20

0 0
0 4 8 12 0 4 8 12
Month Month
Participants at risk, n Participants at risk, n
Vitamin D 91 31 20 4 Vitamin D 91 71 41 16
Placebo 91 30 19 9 Placebo 91 68 38 13

C 100 D 15
Vitamin D Vitamin D
80 Placebo Placebo

Probability of Death
From Any Cause, %
Probability of First
Hospitalization, %

10
60

40
5

20

0 0
0 4 8 12 0 4 8 12
Month Month
Participants at risk, n Participants at risk, n
Vitamin D 91 74 60 37 Vitamin D 91 91 87 82
Placebo 91 71 62 41 Placebo 91 90 88 85

Safety Bacteriology, Plasma Cathelicidin Levels, and

Blood samples were available in 90% of the study vis- Monocyte Phagocytosis
its for the ITT population. Mean serum calcium and phos- Plasma cathelicidin levels were not affected by serum
phate levels did not differ between the groups at any time 25-(OH)D levels or supplementation (Appendix Figure 3,
during follow-up (mean between-group difference in cal- available at www.annals.org). Monocyte capacity for
cium levels, 0.02 mmol/L [0.08 mg/dL] [CI, ⫺0.03 to phagocytosis in the vitamin D group was significantly bet-
0.04 mmol/L {⫺0.01 to 0.16 mg/dL}; P ⫽ 0.061]; mean ter than that in the placebo group (P ⫽ 0.002); this dif-
between-group difference in phosphate levels, ⫺0.006 ference was more pronounced in the subgroup of partici-
mmol/L [⫺0.02 mg/dL] [CI, ⫺0.04 to 0.03 mmol/L pants with severe vitamin D deficiency at baseline
{⫺0.13 to 0.09 mg/dL}; P ⫽ 0.69]) (Appendix Table 3, (Appendix Figure 4, available at www.annals.org). No ef-
available at www.annals.org). At 4 months, 4 cases of fect of vitamin D on the rate of detecting pathogenic
mild and asymptomatic hypercalcemia (defined as serum strains or on the total number of colony-forming units was
calcium levels between 2.63 and 2.75 mmol/L [10.5 and observed (Appendix Table 4, available at www.annals.org).
11.0 mg/dL]) were detected in the vitamin D group
compared with 0 cases in the placebo group (P ⫽
0.043). Despite continuation of the study medication, DISCUSSION
hypercalcemia spontaneously resolved with normal se- This study is, to our knowledge, the first randomized,
rum calcium levels at 8 and 12 months. No cases of placebo-controlled trial to examine the efficacy and safety
hypercalciuria (defined as urinary calcium levels ⬎7.5 of long-term and high-dose vitamin D supplementation in
mmol/d [⬎300 mg/d]) were detected at the end of the COPD. The main finding is that a monthly dose of
study (Appendix Table 3). 100 000 IU of vitamin D in addition to regular therapy
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 High Doses of Vitamin D to Reduce COPD Exacerbations Original Research

Figure 3. Mean serum 25-(OH)D levels and COPD exacerbation rates among the ITT population (A), vitamin D–naive sub-
group (B), and subgroup with severe vitamin D deficiency at baseline (C).

Mean 25-(OH)D Level (95% CI), ng/mL

60
Vitamin D
Placebo
A. ITT Population 50

Variable Vitamin D Placebo Difference or RR P Value 40

(n = 91) (n = 91) (95% CI)

Mean 25-(OH)D 30

level (SD), ng/mL

20
At baseline 20 (12) 20 (11) 0.2 (–3 to 4) 0.90
During study 52 (16) 22 (13) 30 (27 to 33) <0.001 10
COPD exacerbations 2.8 2.9 0.94 (0.76 to 1.16) 0.57
per patient-year, n 0
0 4 8 12
Month
Participants, n
Vitamin D 91 83 79 70
Placebo 91 86 81 74

Mean 25-(OH)D Level (95% CI), ng/mL

60
Vitamin D
Placebo
B. Vitamin D–Naive Subgroup 50

Variable Vitamin D Placebo Difference or RR P Value 40

(n = 71) (n = 71) (95% CI)

Mean 25-(OH)D 30

level (SD), ng/mL

20
At baseline 17 (8) 16 (8) 0.75 (–2 to 3) 0.58
During study 51 (16) 19 (11) 32 (29 to 34) <0.001 10
COPD exacerbations 2.69 2.99 0.88 (0.69 to 1.12) 0.30
per patient-year, n 0
0 4 8 12
Month
Participants, n
Vitamin D 71 65 62 56
Placebo 71 68 67 61
Mean 25-(OH)D Level (95% CI), ng/mL

60
Vitamin D
Placebo
C. Subgroup With Severe Vitamin D Deficiency 50

Variable Vitamin D Placebo Difference or RR P Value 40

(n = 15) (n = 15) (95% CI)

Mean 25-(OH)D 30

level (SD), ng/mL

20
At baseline 8 (2) 7 (2) 0.57 (–1 to 2) 0.36
During study 50 (15) 12 (8) 38 (33 to 44) <0.001 10
COPD exacerbations 1.84 3.45 0.57 (0.33 to 0.98) 0.042
per patient-year, n 0
0 4 8 12
Month
Participants, n
Vitamin D 15 13 12 12
Placebo 15 15 14 13

Two-way analysis-of-variance statistics for between-group differences in serum 25-(OH)D levels and Poisson regression statistics for exacerbation rates are
given. P values are unadjusted. Severe vitamin D deficiency was defined as having serum 25-(OH)D levels ⬍10 ng/mL. To convert values to nmol/L,
multiply by 2.5. 25-(OH)D ⫽ 25-hydroxyvitamin D; COPD ⫽ chronic obstructive pulmonary disease; ITT ⫽ intention-to-treat; RR ⫽ rate ratio.

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 Original Research High Doses of Vitamin D to Reduce COPD Exacerbations

does not reduce the time to first exacerbation or the rate of showed a statistically significant reduction in exacerbations
exacerbations in patients with moderate to very severe of 43% over 1 year. In this subgroup, vitamin D treatment
COPD. Secondary outcomes, such as FEV1, quality of life, was associated with a significant increase in the monocyte
and death, were also not affected. capacity for phagocytosis. Considering the post hoc nature
The absence of a vitamin D–mediated effect in our of the analysis and the small size of the subgroup, therapy
study sample contrasts with indirect evidence from most should not be altered owing to these hypothesis-generating
association studies in COPD (2, 4, 6), but is consistent data. However, because 1 out of 6 participants in the trial
with recent data from the Lung Health Study (30), which presented with such asymptomatic, low serum 25-(OH)D
showed that vitamin levels did not determine the rate of levels at baseline that persisted during the study, further
decline in FEV1 in a limited subgroup. In addition, most focus and future studies on this important subgroup may
intervention trials of vitamin D supplementation targeting be warranted, eventually resulting in better patient-tailored
extracalcemic effects in other chronic diseases have shown interventions. Recently, a frequent exacerbator phenotype
disappointing results (12, 14, 31). Because the absence of was identified suggesting that individualized therapy, in-
clinically significant effects in such trials may relate to a cluding appropriate vitamin D supplementation, may be-
lack of power, insufficient supplementation, or insensitive come important (32). Finally, we should note that the lack
end points, we specifically designed our study to overcome of an overall effect may be explained by local insensitivity
some of these concerns. We carefully assessed the effect of to vitamin D because of smoking or chronic inflammation.
a monthly dose of 100 000 IU of vitamin D on serum Epigenetic silencing of vitamin D signaling has been de-
25-(OH)D levels at several time points during the trial. scribed in several types of cancer, and similar mechanisms
Active treatment resulted in mean serum 25-(OH)D levels may apply to chronic inflammatory diseases, such as
of 52 ng/mL (SD, 16), which is within the therapeutic COPD (36 –38). Of note, we could not detect any differ-
range to obtain the hypothesized extracalcemic effects (15). ential effect of the treatment in former smokers or current
In addition, we selected a study sample prone to severe smokers, with the limitation that the latter group was very
exacerbations (32) by recruiting most (81%) participants small.
during a hospitalization for an acute exacerbation. Conse- Our study also assessed the potential toxicity of pro-
quently, the average number of exacerbations observed (2.9 longed and high-dose supplementation in patients with
per patient-year) was higher than that usually found in COPD, although we acknowledge that it was underpow-
large intervention trials of inhalation therapy in partici- ered to determine long-term safety. Our data demonstrate
pants from an outpatient clinic, thereby enhancing the that an average daily dose of more than 3200 IU of vitamin
power to detect significant differences (33, 34). However, D during 1 year given as once-monthly 100 000 IU, which
the original power calculations, based on a t test to evaluate is more than 4 times the recommended dose for bone pro-
the difference in the mean time to first exacerbation, did tection (39), introduced a small and transient risk for
not consider the skewed distribution of the data on exac- asymptomatic hypercalcemia. Because our blood samples
erbations. Although the limited sample size provided insuf- were collected independently at drug intake, we recognize
ficient power to demonstrate a clinically significant delay in that other transient peaks of hypercalcemia in the days
time to first exacerbation, the survival curves for the time after ingestion may have been missed in some participants.
to first exacerbation almost completely overlapped (HR, This indicates that the upper limit of tolerability of 4000
1.1 [CI, 0.82 to 1.56]) and the exacerbation rate did not IU/d, which is newly recommended and defined by the
differ (rate ratio, 0.94 [CI, 0.76 to 1.16]). Thus, we think Institute of Medicine, may still be too high in elderly, sick
that the probability of making a type II error in our study patients (19).
was rather small. Given the wide interest for vitamin D intervention
The absence of therapeutic effect of vitamin D in our trials in other chronic diseases, these findings may help to
study sample may relate to the fact that most of our par- guide the design of and dosage in future trials. Although
ticipants presented with severe COPD and were receiving our results demonstrate that supplementation beyond what
maximum inhalation therapy. As all of these treatments are is recommended for bone health does not reduce exacerba-
known to reduce exacerbations, any additional effect of tions in patients with moderate to very severe COPD, they
vitamin D in addition to regular treatment is probably corroborate the suggestion that vitamin D deficiency is a
more difficult to obtain (33, 34). Intervention in the earlier potential risk in some patients. The dose used in our study
stages of COPD when patients receive fewer medications was beyond the currently recommended daily requirements
might therefore be more effective, which is consistent with but yielded reliable levels of serum 25-(OH)D in all par-
the idea that such milder stages are also more sensitive to ticipants without any observed symptomatic calcium-
disease modification (35). Second, a post hoc analysis related toxicity. More studies in other chronic diseases are
showed a significant interaction of baseline serum 25- needed to further explore the need and safety for recommend-
(OH)D levels with intervention for exacerbation rates in ing these higher doses of vitamin D to obtain potential ben-
the ITT population. Subsequently, our intervention in par- eficial effects beyond bone health, particularly in vitamin
ticipants with severe vitamin D deficiency (⬍10 ng/mL) D– deficient patients with immune-related diseases.
112 17 January 2012 Annals of Internal Medicine Volume 156 • Number 2 www.annals.org

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 High Doses of Vitamin D to Reduce COPD Exacerbations Original Research
From University Hospitals Leuven, Leuven, Belgium. 14. Wejse C, Gomes VF, Rabna P, Gustafson P, Aaby P, Lisse IM, et al.
Vitamin D as supplementary treatment for tuberculosis: a double-blind, random-
Acknowledgment: The authors thank Laboratoires SMB (Brussels, Bel- ized, placebo-controlled trial. Am J Respir Crit Care Med. 2009;179:843-50.
gium) for providing the vitamin D and placebo solution. [PMID: 19179490]
15. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-81.
[PMID: 17634462]
Grant Support: By the Applied Biomedical Research Program, Agency 16. Vieth R. What is the optimal vitamin D status for health? Prog Biophys Mol
for Innovation by Science and Technology (IWT-TBM) (G.335102) Biol. 2006;92:26-32. [PMID: 16766239]
and Laboratoires SMB (Brussels, Belgium). Drs. Mathieu, Decallonne, 17. Dawson-Hughes B, Mithal A, Bonjour JP, Boonen S, Burckhardt P,
and Janssens are supported by Research Foundation–Flanders (FWO Fuleihan GE, et al. IOF position statement: vitamin D recommendations for
Vlaanderen). older adults. Osteoporos Int. 2010;21:1151-4. [PMID: 20422154]
18. Martineau AR, Timms PM, Bothamley GH, Hanifa Y, Islam K, Claxton
Potential Conflicts of Interest: Disclosures can be viewed at www.acponline AP, et al. High-dose vitamin D(3) during intensive-phase antimicrobial treat-
.org/authors/icmje/ConflictOfInterestForms.do?msNum⫽M11-1083. ment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lan-
cet. 2011;377:242-50. [PMID: 21215445]
19. The National Academies. IOM report sets new dietary intake levels for
Reproducible Research Statement: Study protocol: Synopsis available at
calcium and vitamin D to maintain health and avoid risks associated with excess
ClinicalTrials.gov. Statistical code: Available from Dr. Janssens (e-mail, [press release]. Washington, DC: The National Academies; 30 November 2010.
[email protected]). Data set: Not available. Accessed at www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID
⫽13050 on 5 November 2011.
Requests for Single Reprints: Wim Janssens, MD, PhD, Respiratory 20. Janssens W, Lehouck A, Carremans C, Bouillon R, Mathieu C, Decramer
Medicine, University Hospitals Leuven, Herestraat 49, Bus 7003, 3000 M. Vitamin D beyond bones in chronic obstructive pulmonary disease: time to
Leuven, Belgium; e-mail, [email protected]. act. Am J Respir Crit Care Med. 2009;179:630-6. [PMID: 19164701]
21. Seemungal TA, Wilkinson TM, Hurst JR, Perera WR, Sapsford RJ,
Wedzicha JA. Long-term erythromycin therapy is associated with decreased
Current author addresses and author contributions are available at www
chronic obstructive pulmonary disease exacerbations. Am J Respir Crit Care Med.
.annals.org.
2008;178:1139-47. [PMID: 18723437]
22. Celli BR, Cote CG, Marin JM, Casanova C, Montes de Oca M, Mendez
RA, et al. The body-mass index, airflow obstruction, dyspnea, and exercise ca-
References pacity index in chronic obstructive pulmonary disease. N Engl J Med. 2004;350:
1. Donaldson GC, Seemungal TA, Bhowmik A, Wedzicha JA. Relationship 1005-12. [PMID: 14999112]
between exacerbation frequency and lung function decline in chronic obstructive 23. Charlson M, Szatrowski TP, Peterson J, Gold J. Validation of a combined
pulmonary disease. Thorax. 2002;57:847-52. [PMID: 12324669] comorbidity index. J Clin Epidemiol. 1994;47:1245-51. [PMID: 7722560]
2. Black PN, Scragg R. Relationship between serum 25-hydroxyvitamin d and 24. Roca J, Burgos F, Sunyer J, Saez M, Chinn S, Antó JM, et al. References
pulmonary function in the third national health and nutrition examination sur- values for forced spirometry. Group of the European Community Respiratory
vey. Chest. 2005;128:3792-8. [PMID: 16354847] Health Survey. Eur Respir J. 1998;11:1354-62. [PMID: 9657579]
3. Ross AC. The 2011 report on dietary reference intakes for calcium and vita- 25. Puhan MA, Behnke M, Laschke M, Lichtenschopf A, Brändli O, Guyatt
min D [Letter]. Public Health Nutr. 2011;14:938-9. [PMID: 21492489] GH, et al. Self-administration and standardisation of the Chronic Respiratory
4. Janssens W, Bouillon R, Claes B, Carremans C, Lehouck A, Buysschaert I, Questionnaire: a randomised trial in three German-speaking countries. Respir
et al. Vitamin D deficiency is highly prevalent in COPD and correlates with Med. 2004;98:342-50. [PMID: 15072175]
variants in the vitamin D-binding gene. Thorax. 2010;65:215-20. [PMID: 26. Anthonisen NR, Manfreda J, Warren CP, Hershfield ES, Harding GK,
19996341] Nelson NA. Antibiotic therapy in exacerbations of chronic obstructive pulmo-
5. Lips P. Vitamin D physiology. Prog Biophys Mol Biol. 2006;92:4-8. [PMID: nary disease. Ann Intern Med. 1987;106:196-204. [PMID: 3492164]
16563471] 27. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et
6. Ginde AA, Mansbach JM, Camargo CA Jr. Association between serum 25- al; ATS/ERS Task Force. Standardisation of spirometry. Eur Respir J. 2005;26:
hydroxyvitamin D level and upper respiratory tract infection in the Third Na- 319-38. [PMID: 16055882]
tional Health and Nutrition Examination Survey. Arch Intern Med. 2009;169: 28. Wilson R, Allegra L, Huchon G, Izquierdo JL, Jones P, Schaberg T, et al;
384-90. [PMID: 19237723] MOSAIC Study Group. Short-term and long-term outcomes of moxifloxacin
7. Kriegel MA, Manson JE, Costenbader KH. Does vitamin D affect risk of compared to standard antibiotic treatment in acute exacerbations of chronic
developing autoimmune disease?: a systematic review. Semin Arthritis Rheum. bronchitis. Chest. 2004;125:953-64. [PMID: 15006954]
2011;40:512-531.e8. [PMID: 21047669] 29. Suissa S. Statistical treatment of exacerbations in therapeutic trials of chronic
8. Krishnan AV, Trump DL, Johnson CS, Feldman D. The role of vitamin D obstructive pulmonary disease. Am J Respir Crit Care Med. 2006;173:842-6.
in cancer prevention and treatment. Endocrinol Metab Clin North Am. 2010; [PMID: 16439716]
39:401-18. [PMID: 20511060] 30. Kunisaki KM, Niewoehner DE, Singh RJ, Connett JE. Vitamin D status
9. Nnoaham KE, Clarke A. Low serum vitamin D levels and tuberculosis: a and longitudinal lung function decline in the Lung Health Study. Eur Respir J.
systematic review and meta-analysis. Int J Epidemiol. 2008;37:113-9. [PMID: 2011;37:238-43. [PMID: 20595151]
18245055] 31. Witham MD, Crighton LJ, Gillespie ND, Struthers AD, McMurdo ME.
10. Wang TJ, Pencina MJ, Booth SL, Jacques PF, Ingelsson E, Lanier K, et al. The effects of vitamin D supplementation on physical function and quality of life
Vitamin D deficiency and risk of cardiovascular disease. Circulation. 2008;117: in older patients with heart failure: a randomized controlled trial. Circ Heart Fail.
503-11. [PMID: 18180395] 2010;3:195-201. [PMID: 20103775]
11. Burton JM, Kimball S, Vieth R, Bar-Or A, Dosch HM, Cheung R, et al. A 32. Hurst JR, Vestbo J, Anzueto A, Locantore N, Müllerova H, Tal-Singer R,
phase I/II dose-escalation trial of vitamin D3 and calcium in multiple sclerosis. et al; Evaluation of COPD Longitudinally to Identify Predictive Surrogate
Neurology. 2010;74:1852-9. [PMID: 20427749] Endpoints (ECLIPSE) Investigators. Susceptibility to exacerbation in chronic
12. von Hurst PR, Stonehouse W, Coad J. Vitamin D supplementation reduces obstructive pulmonary disease. N Engl J Med. 2010;363:1128-38. [PMID:
insulin resistance in South Asian women living in New Zealand who are insulin 20843247]
resistant and vitamin D deficient—a randomised, placebo-controlled trial. Br J 33. Calverley PM, Anderson JA, Celli B, Ferguson GT, Jenkins C, Jones PW,
Nutr. 2010;103:549-55. [PMID: 19781131] et al; TORCH investigators. Salmeterol and fluticasone propionate and survival
13. Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. Ran- in chronic obstructive pulmonary disease. N Engl J Med. 2007;356:775-89.
domized trial of vitamin D supplementation to prevent seasonal influenza A in [PMID: 17314337]
schoolchildren. Am J Clin Nutr. 2010;91:1255-60. [PMID: 20219962] 34. Tashkin DP, Celli B, Senn S, Burkhart D, Kesten S, Menjoge S, et al;

www.annals.org 17 January 2012 Annals of Internal Medicine Volume 156 • Number 2 113

Downloaded From: http://annals.org/ on 07/03/2014

 Original Research High Doses of Vitamin D to Reduce COPD Exacerbations

UPLIFT Study Investigators. A 4-year trial of tiotropium in chronic obstructive cer Biol Ther. 2010;10:44-53. [PMID: 20431345]
pulmonary disease. N Engl J Med. 2008;359:1543-54. [PMID: 18836213] 38. Essa S, Denzer N, Mahlknecht U, Klein R, Collnot EM, Tilgen W, et al.
35. Decramer M, Cooper CB. Treatment of COPD: the sooner the better? VDR microRNA expression and epigenetic silencing of vitamin D signaling in
Thorax. 2010;65:837-41. [PMID: 20805184] melanoma cells. J Steroid Biochem Mol Biol. 2010;121:110-3. [PMID:
36. Ito K, Ito M, Elliott WM, Cosio B, Caramori G, Kon OM, et al. Decreased 20153427]
histone deacetylase activity in chronic obstructive pulmonary disease. N Engl J 39. Bischoff-Ferrari HA, Willett WC, Wong JB, Stuck AE, Staehelin HB, Orav
Med. 2005;352:1967-76. [PMID: 15888697] EJ, et al. Prevention of nonvertebral fractures with oral vitamin D and dose
37. Marik R, Fackler M, Gabrielson E, Zeiger MA, Sukumar S, Stearns V, et al. dependency: a meta-analysis of randomized controlled trials. Arch Intern Med.
DNA methylation-related vitamin D receptor insensitivity in breast cancer. Can- 2009;169:551-61. [PMID: 19307517]

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 Annals of Internal Medicine
Current Author Addresses: Dr. Lehouck: Respiratory Medicine, University counted at their respective dilution in plates on which 1 to ap-
Hospitals Leuven, UZ Herestraat 49, Bus 7003, 3000 Leuven, Belgium. proximately 200 colonies could be differentiated.
Drs. Mathieu, Baeke, Decallonne, and Bouillon: Endocrinology and Ex-
perimental Medicine, University Hospitals Leuven, O&N I Herestraat
49, Bus 902, 3000 Leuven, Belgium. Monocyte Phagocytosis Assay
Ms. Carremans and Dr. Janssens: Respiratory Medicine, University Hos- The phagocytosis assay was performed by using the Phagotest
pitals Leuven, Herestraat 49, Bus 7003, 3000 Leuven, Belgium. Kit (ORPEGEN Pharma, Germany) containing fluorescein isothio-
Drs. Verhaegen and Van Eldere: Laboratory of Bacteriology, University cyanate (FITC)–labeled, opsonized Escherichia coli. Samples of 100
Hospitals Leuven, UZ Herestraat 49, Bus 7003, 3000 Leuven, Belgium. ␮L of peripheral blood with heparin were cooled on ice for 15
Dr. Decramer: Respiratory Medicine, University Hospitals Leuven, UZ
minutes, mixed with 2 ⫻ 107 FITC-labeled E. coli, and subse-
Herestraat 49, Bus 7003, 3000 Leuven, Belgium.
quently placed at 37 °C for 10 minutes. Control samples were kept
Author Contributions: Conception and design: C. Mathieu, C. Carre- on ice to inhibit phagocytosis. Next, 100 ␮L of brilliant blue
mans, F. Baeke, J. Van Eldere, B. Decallonne, R. Bouillon, W. Janssens. (quenching solution) was added to delete fluorescence of nonphago-
Analysis and interpretation of the data: A. Lehouck, C. Mathieu, F. cytosed bacteria sticking to the cellular membrane. After 2 washing
Baeke, B. Decallonne, R. Bouillon, M. Decramer, W. Janssens. steps, erythrocytes were lysed with lysis buffer for 20 minutes at
Drafting of the article: A. Lehouck, C. Mathieu, F. Baeke, J. Verhaegen, W. room temperature. Three washing steps later, 50 ␮L of propidium
Janssens.
iodide was added to stain leukocytes and intracellular bacterial
Critical revision of the article for important intellectual content: C. Ma-
thieu, F. Baeke, B. Decallonne, R. Bouillon, M. Decramer, W. Janssens.
DNA. The percentage of phagocytosis-positive cells in the monocyte
Final approval of the article: C. Mathieu, F. Baeke, B. Decallonne, R. gate was assessed by flow cytometry.
Bouillon, M. Decramer, W. Janssens. Results
Provision of study materials or patients: C. Mathieu, C. Carremans, J. Exacerbations and Serum 25-(OH)D Levels in the
Verhaegen, W. Janssens.
On-Treatment Population
Statistical expertise: A. Lehouck, W. Janssens.
Obtaining of funding: C. Mathieu, M. Decramer, W. Janssens.
Kaplan–Meier survival analysis showed no significant differ-
Administrative, technical, or logistic support: C. Carremans, F. ence in the median time to first exacerbation between the vitamin
Baeke, J. Van Eldere. D group and the placebo group (HR, 1.15 [CI, 0.83 to 1.60];
Collection and assembly of data: A. Lehouck, C. Mathieu, C. Carre- P ⫽ 0.39) (Appendix Figure 1, top). Supplementation with vita-
mans, F. Baeke, J. Verhaegen, J. Van Eldere, W. Janssens. min D resulted in a steep and significant increase in serum 25-
(OH)D levels that remained stable during the study (mean, 52
APPENDIX ng/mL [SD, 16]), whereas baseline levels in the placebo group
Methods remained stable during the study (mean between-group differ-
Serum 25-(OH)D Levels ence, 30 ng/mL [CI, 27 to 33 ng/mL]; P ⬍ 0.001) (Appendix
Total serum 25-(OH)D levels were measured in multiple Figure 1, middle). No difference in the annual rate of exacerba-
batches by radioimmunoassay (DiaSorin, Brussels, Belgium) in all tions was observed (P ⫽ 0.99) (Appendix Figure 1, bottom).
study participants according to the standard protocol. They are
mean values of duplicate measures. Levels are expressed in nano- Lung Function
grams per liter (conversion factor for nanomoles per liter, 2.5). Spirometric data from the study visits at baseline and at 4, 8,
and 12 months were analyzed for trends over time in FEV1. In
Plasma Cathelicidin Levels 89% of the visits in the ITT population, appropriate FEV1 mea-
Plasma cathelicidin levels were assayed by using an enzyme- surements were obtained. Linear mixed-model analysis showed
linked immunosorbent assay (Hycult Biotechnology, Uden, the no significant differences in FEV1 between the vitamin D and
Netherlands), according to the standard protocol. placebo groups, nor among study visits (P ⫽ 0.87) (Appendix
Figure 2 and Appendix Table 1).
Quantitative Bacterial Cultures
Participants were asked to collect a spontaneous morning Quantitative Bacterial Cultures
sputum on the day of a study visit. A cooling box to transport the The nature of spontaneous morning sputa cultures did not signif-
sample was provided, and participants were instructed to blow icantly differ for pathogenic strains or for the total number of colony-
their nose and rinse their mouth with water before collecting forming units when comparing both groups (Appendix Table 4).
sputum. Sputum was homogenized by adding 0.1% dithiothrei-
tol, and serial dilutions were prepared in phosphate-buffered sa- Monocyte Capacity for Phagocytosis
line at 1:100, 1:1000, 1:10 000, and 1:100 000. These dilutions Monocyte function evaluated at the end of the study showed
were cultured on chocolate agar, blood agar, MacConkey agar, a significantly better capacity for phagocytosis in the vitamin D
and mannitol salt agar plates. Undiluted but homogenized spu- group than in the placebo group (P ⫽ 0.002) (Appendix Figure
tum samples were also cultured on chocolate agar and blood agar 4, top). The observed difference was more pronounced in the
plates. Colonies of bacteria were examined and identified after subgroup of participants with severe vitamin D deficiency at
24-hour incubation at 37 °C. Colony-forming units were baseline (P ⫽ 0.002) (Appendix Figure 4, bottom).

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 Appendix Figure 1. Mean serum 25-(OH)D levels and COPD Appendix Figure 2. FEV1 from baseline to end of study in the
exacerbation rates among the on-treatment population. intention-to-treat population.

1.6
Probability of First Exacerbation, %

100 Vitamin D Vitamin D

Placebo 1.5 Placebo
75

Mean FEV1 (95% CI), L

1.4

1.3
50

1.2

25 1.1

1.0
0
0 4 8 12 0.9
Month
Participants at risk, n 0.8
0 4 8 12
Vitamin D 91 29 17 4
Month
Placebo 91 29 18 9 Participants, n
Vitamin D 91 81 76 69
Mean 25-(OH)D Level (95% CI), ng/mL

60 Vitamin D Placebo 91 84 81 77
Placebo
50 Linear mixed-model analysis showed no significant differences between
the groups and study visits (P ⫽ 0.87).
40

30

20

10

0
0 4 8 12
Month
Participants, n
Vitamin D 91 81 75 65
Placebo 91 83 73 65

Variable Vitamin D Placebo Difference or RR P Value

(n = 91) (n = 91) (95% CI)

Mean 25-(OH)D
level (SD), ng/mL
At baseline 20 (12) 20 (11) 0.2 (–3 to 4) 0.90
During study 52 (16) 22 (13) 30 (27 to 33) <0.001
COPD exacerbations 2.8 2.8 0.99 (0.80 to 1.25) 0.99
per patient-year, n
Two-way ANOVA <0.001

25-(OH)D ⫽ 25-hydroxyvitamin D; ANOVA ⫽ analysis of variance;

COPD ⫽ chronic obstructive pulmonary disease; RR ⫽ rate ratio. Top.
Kaplan–Meier plots of time to first exacerbation in the on-treatment
population. Middle. Mean serum 25-(OH)D levels in the on-treatment
population, excluding patients who started low-dose vitamin D supple-
mentation during the trial. Bottom. COPD exacerbations per patient-
year in the on-treatment population. Two-way ANOVA statistics for
between-group differences in serum 25-(OH)D levels and interaction are
given. For exacerbation rates, Poisson regression statistics are given. P
values are unadjusted.

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 Appendix Table 1. Serial Measurements of Lung Function and Quality of Life by Chronic Respiratory Questionnaire Scores Over 1
Year in the Intention-to-Treat Population*

Variable Vitamin D Group Placebo Group Difference (95% CI) Unadjusted

(n ⴝ 91) (n ⴝ 91) P Value
FEV1, L
Baseline 1.2 (0.4) 1.2 (0.4) 0.05 (⫺0.08 to 0.17) 0.49
During study 1.2 (0.5) 1.2 (0.5) ⫺0.01 (⫺0.09 to 0.08) 0.95
P value† 0.88

Dyspnea score
Baseline 4.7 (1.4) 4.7 (1.5) ⫺0.03 (⫺0.46 to 0.40) 0.90
During study 4.8 (1.5) 5.0 (1.5) ⫺0.24 (⫺0.49 to 0.14) 0.141
P value† 0.30

Emotional score
Baseline 4.9 (1.3) 4.8 (1.3) 0.10 (⫺0.27 to 0.47) 0.60
During study 5.0 (1.3) 5.1 (1.3) ⫺0.07 (⫺0.31 to 0.16) 0.55
P value† 0.53

Fatigue score
Baseline 4.1 (1.4) 3.9 (1.4) 0.19 (⫺0.22 to 0.59) 0.36
During study 4.3 (1.5) 4.1 (1.4) 0.15 (⫺0.12 to 0.42) 0.27
P value† 0.27

Mastery score
Baseline 5.3 (1.3) 5.2 (1.3) 0.09 (⫺0.28 to 0.48) 0.63
During study 5.4 (1.3) 5.5 (1.3) ⫺0.02 (⫺0.26 to 0.22) 0.85
P value† 0.57

* Values are means (SDs).

† Two-way analysis-of-variance P value for interaction.

Appendix Table 2. Serum 25-(OH)D Levels Over 1 Year in the ITT Population, Subgroup of Vitamin D–Naive Participants, and
Subgroup of Vitamin D–Deficient Participants*

Variable† Vitamin D Group Placebo Group Difference (95% CI) Unadjusted P Value
ITT population
Baseline (n ⫽ 91/91) 20 (12) 20 (11) 0.2 (–3 to 4) 0.90
4 mo (n ⫽ 83/86) 52 (13) 23 (13) 28 (25 to 32) ⬍0.001
8 mo (n ⫽ 79/81) 54 (15) 23 (12) 31 (27 to 35) ⬍0.001
12 mo (n ⫽ 70/74) 52 (19) 20 (13) 32 (26 to 37) ⬍0.001
During study 52 (16) 22 (13) 30 (27 to 33) ⬍0.001
P value‡ ⬍0.001

Vitamin D–naive subgroup

Baseline (n ⫽ 71/71) 17 (8) 16 (8) 0.8 (–2 to 3) 0.58
4 mo (n ⫽ 65/68) 51 (14) 20 (11) 31 (26 to 35) ⬍0.001
8 mo (n ⫽ 62/67) 52 (16) 20 (10) 32 (27 to 36) ⬍0.001
12 mo (n ⫽ 56/61) 50 (18) 18 (10) 33 (27 to 38) ⬍0.001
During study 51 (16) 19 (11) 32 (29 to 34) ⬍0.001
P value‡ ⬍0.001

Vitamin D–deficient subgroup

Baseline (n ⫽ 15/15) 8 (2) 7 (2) 0.6 (–1 to 2) 0.36
4 mo (n ⫽ 13/15) 49 (18) 15 (10) 34 (22 to 45) ⬍0.001
8 mo (n ⫽ 12/14) 51 (13) 12 (8) 39 (31 to 48) ⬍0.001
12 mo (n ⫽ 12/13) 50 (13) 8 (3) 43 (35 to 50) ⬍0.001
During study 50 (15) 12 (8) 38 (33 to 44) ⬍0.001
P value‡ ⬍0.001

25-(OH)D ⫽ 25-hydroxyvitamin D; ITT ⫽ intention-to-treat.

* Values are means (SDs), reported in ng/mL. To convert values to nmol/L, multiply by 2.5.
† The numerator and denominator represent the numbers of participants in the vitamin D/placebo groups.
‡ Two-way analysis-of-variance P value for interaction.

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 Appendix Table 3. Safety: Serial Serum Calcium and Phosphate Levels and Incidence of Hypercalcemia and Hypercalciuria Over 1
Year in Vitamin D and Placebo Groups in the Intention-to-Treat Population*

Variable† Vitamin D Group Placebo Group Difference (95% CI) P Value

Calcium level, mg/dL‡
Baseline (n ⫽ 91/91) 9.4 (0.50) 9.4 (0.37) 0.04 (⫺0.09 to 0.16) 0.59
4 mo (n ⫽ 83/86) 9.4 (0.50) 9.3 (0.42) 0.07 (⫺0.07 to 0.21) 0.32
8 mo (n ⫽ 79/81) 9.3 (0.38) 9.3 (0.42) 0.05 (⫺0.07 to 0.18) 0.39
12 mo (n ⫽ 70/74) 9.3 (0.41) 9.2 (0.44) 0.11 (⫺0.04 to 0.26) 0.146
During study 9.4 (0.43) 9.3 (0.43) 0.08 (⫺0.01 to 0.16) 0.061
P value§ 0.86

Phosphate level, mg/dL㥋

Baseline (n ⫽ 91/91) 2.8 (0.59) 2.8 (0.53) ⫺0.03 (⫺0.20 to 0.13) 0.70
4 mo (n ⫽ 83/86) 2.8 (0.61) 2.8 (0.60) 0.01 (⫺0.18 to 0.20) 0.93
8 mo (n ⫽ 79/81) 2.8 (0.57) 2.9 (0.59) ⫺0.06 (⫺0.25 to 0.12) 0.50
12 mo (n ⫽ 70/74) 2.9 (0.51) 2.9 (0.56) ⫺0.01 (⫺0.20 to 0.17) 0.90
During study 2.8 (0.57) 2.9 (0.58) ⫺0.02 (⫺0.13 to 0.09) 0.69
P value§ 0.95

Hypercalcemia, % (n)
Baseline (n ⫽ 91/91) 1 (2) 0 – 0.155
4 mo (n ⫽ 83/86) 2 (4) 0 – 0.043
8 mo (n ⫽ 79/81) 0 0 – –
12 mo (n ⫽ 70/74) 0 0 – –

Hypercalciuria, % (n)¶
Baseline – – – –
4 mo – – – –
8 mo – – – –
12 mo (n ⫽ 39/40) 0 0 – –

* Values are reported as means (SDs).

† The numerator and denominator represent the numbers of participants in the vitamin D/placebo groups.
‡ To convert calcium values to mmol/L, multiply by 0.25.
§ Two-way analysis-of-variance P value for interaction.
㛳 To convert phosphate values to mmol/L, multiply by 0.323.
¶ The presence of hypercalciuria was examined only at the end of the study.

Appendix Table 4. Sputum Samples and Bacterial Cultures

Variable Baseline 4 mo 8 mo 12 mo
Participants providing spontaneous sputum samples, % (n)
Vitamin D group (n ⫽ 91) 54 (49) 44 (40) 33 (30) 20 (18)
Placebo group (n ⫽ 91) 49 (45) 47 (43) 44 (40) 30 (28)
P value* 0.55 0.66 0.128 0.088

Sputum samples with a positive culture, % (n/n)†

Vitamin D group (n ⫽ 91) 26 (11/49) 28 (11/40) 40 (12/30) 28 (5/18)
Placebo group (n ⫽ 91) 52 (23/45) 47 (20/43) 45 (18/40) 32 (9/28)
P value* 0.011 0.074 0.68 0.75

Positive samples, % (n)

Vitamin D group
0 CFUs/mL (no potential pathogen) 74 (38) – – 72 (13)
105–106 CFUs/mL 7 (3) – – 0
106–107 CFUs/mL 3 (1) – – 0
107–108 CFUs/mL 7 (3) – – 11 (2)
⬎108 CFUs/mL‡ 9 (4) – – 17 (3)
Placebo group
0 CFUs/mL (no potential pathogen) 48 (22) – – 67 (19)
105–106 CFUs/mL 7 (3) – – 4 (1)
106–107 CFUs/mL 18 (8) – – 4 (1)
107–108 CFUs/mL 9 (4) – – 11 (3)
⬎108 CFUs/mL§ 18 (8) – – 14 (4)

CFU ⫽ colony-forming unit.

* P values express differences between rows according to chi-square statistics.
† Bacterial growth of upper airway commensal flora was not taken into account.
‡ P ⫽ 0.056 between groups.
§ P ⫽ 0.85 between groups.

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 Appendix Figure 3. Plasma cathelicidin levels. Appendix Figure 4. Monocyte phagocytosis assay results.

100 P = 0.002
100
90
90
Cathelicidin Level, ng/mL

80
80

Phagocytosis-Positive in
70

the ITT Population, %

Mean Baseline

60 70

50 60
40 50
30 40
20 30
10
20
0
10
0 10 20 30 40 50 60
Mean Baseline 25-(OH)D Level, ng/mL 0
Vitamin D Placebo
20
(n = 57) (n = 61)
Vitamin D
During Study in ITT Population, ng/mL

18 Placebo P = 0.002
100

Phagocytosis-Positive in the Subgroup

With Severe Vitamin D Deficiency, %
Mean Cathelicidin Level

16
90

14 80

70
12
60
10
50

8 40

30
6
20
4
10
0 4 8 12
Month 0
Participants, n Vitamin D Placebo
Vitamin D 74 70 66 60 (n = 10) (n = 10)

Placebo 76 70 69 63
ITT ⫽ intention-to-treat. Top. Percentage of monocytes positive for
phagocytosis at the end of the study in the ITT population. Bottom.
25-(OH)D ⫽ 25-hydroxyvitamin D; ITT ⫽ intention-to-treat. Top. Percentage of monocytes positive for phagocytosis at the end of the study
Relationship between plasma cathelicidin and serum 25-(OH)D levels at in the subgroup of participants with severe vitamin D deficiency (serum
baseline. Spearman r ⫽ 0.027; P ⫽ 0.04. Bottom. Plasma cathelicidin 25-hydroxyvitamin D levels ⬍10 ng/mL).
levels during the study in the ITT population. Linear mixed-model anal-
ysis showed no significant differences between the groups and study visits
(P ⫽ 0.85).

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