Journal of Pediatric Gastroenterology and Nutrition, Publish Ahead of Print

DOI : 10.1097/MPG.0000000000002126

Increasing Vitamin D Serum Levels Is Associated with Reduced Pulmonary Exacerbations

in Patients with Cystic Fibrosis

Yasmeen Abu-Fraiha1, M.D

Hila Elyashar-Earon1, M.Sc

David Shoseyov1, M.D

Malena Cohen-Cymberknoh1, M.D

Shoshana Armoni1, M.Sc

Prof. Eitan Kerem1, M.D

Prof. Michael Wilschanski1, M.D

1
CF Center, Hadassah-University Medical Center, Jerusalem, Israel

Precis: Supplementing high doses of vitamin D in 90 cystic fibrosis patients for 1-2 years
showed an association between improved vitamin D levels and a decrease in pulmonary
exacerbations.

Abbreviated Title:

Increased Vitamin D Reduces Pulmonary Exacerbations in CF

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Correspondence:

Dr. Yasmeen Abu-Fraiha

18th Hakeremst., Omer

POB 1230, Zip code 8496500

Israel

+972-505-607045

yasmeenaf23@gmail.com

Disclosure: There are no conflicts of interest to disclose. This research did not receive any
financial support, including grants.

ClinicalTrials.gov Identifier: NCT02043717

Other Study ID Numbers:vitaminD-HMO-CTIL

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ABSTRACT

Context: In 2012, The North American CF Foundation published new guidelines for the

treatment of vitamin D deficiency in individuals with CF.

Objective: The objectives of our study were to assess the efficacy of these guidelines, and to test

the effect of increasing vitamin D dosage on pulmonary function and exacerbations.

Design: Pulmonary function tests and serum concentrations of 25-hydroxyvitamin D [25(OH)D]

were measured one year prior to increasing vitamin D dosage according to the guidelines and at

least one year later. In addition, days of hospitalization (DOH) and pulmonary exacerbations (PE)

were counted and an average per year (DOHA and PEA, respectively) was calculated.

Setting and Participants: 90 patients from The CF Clinic at Hadassah Mount-Scopus Hospital,

Jerusalem, Israel.

Results: The mean serum concentration of vitamin D increased significantly from 20.97 ng/ml

(52.34 nmol/L) at baseline to 25.41 ng/ml (63.42 nmol/L) at the end of follow-up (p<0.001). The

number of PEA decreased significantly from 2.79±3.96 to 2.15±2.91 (p=0.007). The change in

vitamin D levels was correlated with a decrease in PEA (correlation coefficient = -0.318,

p=0.002).

Conclusions: The NACFF guidelines for management of vitamin D deficiency improve vitamin

D levels in patients with CF but did not reach the normal values in most patients. However, the

increase in vitamin D serum levels was associated with a decrease in number of pulmonary

exacerbations.

Keywords: Vitamin D, Cystic Fibrosis, Pulmonary function, Hospitalizations

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What is known?

• Vitamin D deficiency is a common problem among CF patients.

• Several supplementation protocols have been recommended, with no significant success.

In 2012, the North American CF Foundation, published new guidelines.

• Vitamin D seems to be important in improving pulmonary function in CF patients.

What is new?

• The new North American CF Foundation guidelines are still not sufficient to normalize

vitamin D levels.

• Increasing serum vitamin D levels were associated with a decrease in number of

respiratory exacerbations.

• Pulmonary function and number of hospitalization days did not change.

• This finding may be cost-effective in the long term.

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 1. INTRODUCTION

Vitamin D is a pro-hormone that increases calcium absorption in the bowel, inhibits its

urine secretion, and enhances bone matrix mineralization that prevents rickets in children and

osteomalacia in adults. There has been a change in the perception of vitamin D deficiency in

patients with Cystic Fibrosis (CF). New studies show that vitamin D deficiency is a common

problem among patients(1,2). Several different supplementation protocols have been

recommended in order to correct vitamin D levels in CF, with no significant success(1,3–5).

In addition, there is increasing evidence regarding vitamin D importance in improving

pulmonary function in CF patients(1), in patients with other chronic lung diseases(6), and in

healthy subjects(7). Black et al.(7) demonstrated a dose-response relationship between serum

vitamin D levels and forced expiratory volume in the 1st second (FEV1) and forced vital capacity

(FVC). Grossman et al.(8)demonstrated an association between high dose of vitamin D

supplementation for CF patients with pulmonary exacerbations and a decrease in pro-

inflammatory factors such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. There are also

studies that demonstrate presence of vitamin D receptor (VDR) in the lungs(9), with an unknown

role, and an anti-inflammatory function of the vitamin(10). These results are supported by animal

model research that show an association between vitamin D and pulmonary diseases(10–12).

In 2002, Borowitz et al.(13)published guidelines recommending supplementation of

vitamin D deficient patients with a once weekly dose of 50,000 IU ergocalciferol in patients >5

years and 12,000 IU in patients <5 years of age, for a period of 8 weeks. The normal range of

serum vitamin levels was defined as 30-60 ng/ml (75-150 nmol/L), and patients with vitamin D

levels below 30 ng/ml (75 nmol/L) were defined as vitamin D deficient. However, subsequent

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studies have demonstrated that this protocol was not satisfactory in maintaining vitamin D levels

above this cutoff in most patients(1,3). In 2007, other guidelines were published(14) and

determined that vitamin D levels should be measured annually, recommending that vitamin D

deficient patients should be supplemented with 400 IU ergocalciferol in infants, 400-800 IU in

patients <12 years old, and 2,000 IU/day in patients >12 years. This protocol was also found to be

unsuccessful, and even a 4.5-fold higher dosage failed to increase vitamin D levels to the normal

range(4).

In 2012, the North American CF Foundation, published new guidelines(15), which

included a detailed protocol for supplementing vitamin D in patients with CF. The aim of this

study was to assess the efficacy of these new guidelines to normalize vitamin D levels, and to test

the effect of increasing vitamin D dosage on pulmonary function and pulmonary exacerbations.

2. METHODS

The study population included patients with CF from the CF Center in the Pediatric

Center for Chronic Diseases at Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Inclusion criteria included [1] a definite genetic diagnosis of CF; [2] a complete medical and

nutritional follow-up for at least one year before changing the vitamin D dosage for the first time

according to the NACFF guidelines; and [3] follow up for at least one year afterwards. During the

study period, from June 2012 until December 2014, vitamin D dosage was adjusted for all

subjects according to the CF Foundation guidelines. The follow-up period for each subject started

in the first dosage change according to the guidelines. The study was approved by the Hadassah

Medical Center ethics committee and all patients or parents gave their informed consent to

participate in the study.

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 Baseline characteristics including age, sex, body mass index (BMI) and exocrine

pancreatic status were recorded. In addition, the subjects, or their parents, were asked to report

their own compliance to medical treatment as good compliance (GC) or poor compliance (PC).

That is, compliance was defined only by self-report and it was not specific to vitamin D

supplementation. Serum levels for 25(OH)D were measured using a standardized clinical assay,

the DiaSorin LIAISON 25-OH vitamin D TOTAL. Calcium and phosphorus were also measured.

Pulmonary function tests (PFTs) were recorded by spirometry in patients older than 4 years who

were able to perform the tests.

The number of days of hospitalization (DOHs) and pulmonary exacerbations (PEs) were

recorded in the two years preceding the change in vitamin D dosage as appeared in the patient

records. DOH was defined as a day of admission to the hospital due to a CF-related complication

such as pulmonary exacerbation, intravenous (IV) antibiotic treatment, pancreatitis, distal

intestinal obstruction syndrome (DIOS), constipation and gastrointestinal bleeding or IV

antibiotic treatment at home or as an outpatient at the CF Center. A PE was defined according to

the research definition of the EuroCare CF Working Group(16) – the need for additional

antibiotic treatment as indicated by a recent change in at least two of the following: change in

sputum volume, color or consistency, increased cough, increased malaise, fatigue or lethargy,

increased dyspnea, anorexia or weight loss, decrease in FEV1by ≥10% and/or presence of

radiographic changes. The average number of DOHs and PEs per year were calculated (DOHA

and PEA, respectively).

After establishing baseline values, vitamin D was supplemented according to the CF

Foundation guidelines as follows:

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1. Maintain a serum 25(OH)D goal of at least 30 ng/ml ( ≥ 75 nmol/L). Serum 25(OH)D was

rechecked 3 months after the dose of vitamin D3 has been changed.

2. All individuals with CF, age between 12 months to 10 years, received an initial dose of

800-1,000 IU vitamin D3 (cholecalciferol) / day. If serum 25(OH)D levels were between

20 to 30 ng/ml (50-75 nmol/L), the dose of vitamin D3 was increased to 1,600-3,000 IU /

day. If serum 25(OH)D levels were <20 ng/ml (<50 nmol/L) or with persistent serum

25(OH)D levels of 20-30 ng/ml (50-75 nmol/L) and with confirmed adherence to the

prescribed regimen, the dose of vitamin D3 was increased to a maximum of 4,000 IU per

day. If serum 25(OH)D levels of at least 30 ng/ml ( ≥ 75 nmol/L) was not achieved,

patients with 4,000 IU vitamin had a consultation with a specialist in vitamin D therapy

and higher doses were given.

3. Patients over the age of 10 years were treated with an initial dose of 800-2,000 IU vitamin

D3/day. If the serum 25(OH)D levels were 20-30 ng/ml (50-75 nmol/L), the dose of

vitamin D3 was increased to 1,600-6,000 IU per day. If the serum 25(OH)D levels were

<20 ng/ml (50 nmol/L) or with a persistent serum 25(OH)D levels of 20-30 ng/ml (50-70

nmol/L) despite good adherence, the dose of vitamin D3 was increased to a maximum of

10,000 IU per day. If the serum 25(OH)D levels did not reach at least 30 ng/ml ( ≥ 75

nmol/L) after treatment with 10,000 IU vitamin D3 per day, and with confirmed adherence

to the prescribed regimen, a consultation with a specialist in vitamin D therapy was

provided.

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 25(OH)D was measured again one year later(15). All subjects were supplemented with

oral cholecalciferol, mostly as AquaADEKs® with complementary cholecalciferol drops if

needed.

The baseline measurements were held as part of the CF Clinic regular schedule, and as

such they were measured in different months of the year for different subjects. The follow-up

measurements, 12 months later, took place in the same month of the year in which baseline

measurements were taken, since vitamin D levels change seasonally. Also, this study did not

interfere with other chronic treatments, that were given according to the respective guidelines.

There was no change in dosing of Creon or other supplements during the time frame of the study.

Pearson's correlation coefficient was used in order to test the correlation between two

quantitative variables (for example, between vitamin D levels and average number of pulmonary

exacerbations). The association itself was tested by χ2 test or Fisher's exact test. An association

between a quantitative variable and a qualitative variable (for example, between vitamin D levels

and compliance) was analyzed by T-test.

The variables that were found significantly associated to a dependent quantitative variable

(for example, FEV1) were inserted to a multi-variable model of analysis of covariance. When the

dependent variable was dichotomous (for example, poor compliance vs. good compliance),

logistic regression was used. Wilcoxon signed-rank test was used for variables that had a skewed

distribution (e.g. DOHA) in our study population.

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 3. RESULTS

Ninety subjects were included in the study, with average duration of follow-up was 16.99

months (median 16.28, SD 5.62). Their baseline characteristics are outlined in Table 1. Table 2

displays the variable baseline characteristics compared to the measures at the end of follow-up.

The number of patients with normal vitamin D levels increased from 20 patients (22.2%)

at baseline to 27 patients (30%) at the end of follow-up. The number of patients with vitamin D

levels of <30 ng/ml (<75 nmol/L) but ≥20 ng/ml (≥50 nmol/L) increased from 25 (27.8%)

patients at baseline to 39 (43.3%) patients at the end of the study, and the number of patients with

vitamin D levels of <20 ng/ml (<50 nmol/L) decreased from 45 (50%) patients at baseline to 24

(26.7%) patients at the end of the study. There was no significant difference between the number

of PEA (p=0.6) and DOHA (p=0.2) between the groups, possibly because of the small number of

patients in each group (a minimum of 70 patients each is required to show significance).

There was a significant decrease in PEA (p=0.007) but not in DOHA (p=0.26). In the

two years before changing vitamin D dosage, 51 (57%) of patients were hospitalized (mean and

median DOHA were 20.00 and 5.00, respectively, SD 33.02) while in the two years after starting

the new protocol, 45 (50%) of the patients were hospitalized (mean and median DOHA were

19.04 and 0.5, respectively, SD 40.48). Likewise in the two years before changing vitamin D

dosage, 74 (82%) of patients had at least one pulmonary exacerbation (mean PEA = 2.79, median

= 1.5, SD = 3.96) whereas in the two years after starting the new protocol, 72 (80%) of the

patients had at least one pulmonary exacerbation (mean PEA = 2.15, median = 1.5, SD = 2.91).

There was no change on FEV1 before and after starting the new protocol (Table 2).

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 An inverse association was found between 25(OH)D levels and DOHA (correlation

coefficient = -0.484, p<0.001; Figure 1A) and between 25(OH)D levels and PEA (correlation

coefficient = -0.318, p=0.002; Figure 1B and Table 3).

4. DISCUSSION

This study demonstrates that the North American CF Foundation guidelines for vitamin D

supplementation were associated with an increase in serum vitamin levels. However, they did not

reach the desired serum levels in most patients. The probable reasons include poor compliance to

treatment, malabsorption due to pancreatic insufficiency and insufficient dose. Hence, some

patients needed megadoses of vitamin D (>10,000 IU) in order to increase their serum levels.

Another reason for not reaching the desired levels is the lack of clarity whether patterns of

vitamin D metabolism are the same as those in healthy controls. Thus, further research is needed

to measure other vitamin D metabolites, such as calcitriol and 24,25-dihydroxyvitamin D, that

were not measured in this study, in order to determine if the low elevation of serum 25(OH)D is

due to metabolic dysregulation.

Poor compliance to medical therapy is a well known problem among CF patients.

Nonetheless, our results do not demonstrate a significant difference between patients with GC

and those with PC. The explanations for this finding vary from true lack-of-difference, because

of a possibly good efficacy of the new protocol, to a small sample size that allows equivocal

conclusions.

This is the first study that tests the efficacy of the new protocol, and it supports other

studies that show a need of much higher doses of vitamin D(17,18) in order to reach the desired

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level of ≥ 30 ng/ml (75 nmol/L). Recently, Coriati et al. (18) suggested a new protocol that uses

the combination of a yearlong daily multivitamin pill with a daily specific vitamin D

supplementation, which is doubled during the winter period. They showed a significant increase

in the percentage of patients with 25(OH)D levels equal or greater than 30 ng/ml ( ≥ 75 nmol/L),

and this finding should be further explored.

In addition, the results demonstrate a significant correlation between the increase in

vitamin D level and PEA, similar to previous research(19,20). Importantly, although there is a

well known correlation between PFTs and PEA, this study did not find a correlation between

FEV1 and vitamin D level but did find a significant correlation to FEV1/FVC. This issue is still

not settled in the available literature and many contradicting studies were published on this

subject(1,3,4,7,19,21). However, the significant decrease in PEA in this study cannot be

explained by changes in PFTs.

Pulmonary exacerbations were shown to be an important cause of FEV1 decline in

CF(22). Decreasing the number of pulmonary exacerbations by simply raising serum vitamin D

levels, as demonstrated in this study, may improve the course of the disease and patients' quality

of life. The significance of this finding may be cost-effective, as it was also associated with a

decrease in hospitalization and the costs of treatment for exacerbations, reduces absence from

school and work, increases life expectancy and improves quality of life.

The connection between vitamin D and pulmonary infections is recently being more

recognized. Other studies show similar results(17,18,20) that can be explained by the potential

role of vitamin D in maintaining immune homeostasis and preventing development of

autoimmune processes(23), and by the ability of respiratory epithelial cells to convert inactive

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25(OH)D to active 1,25(OH)2D3 and synergistically help the immune system(24). It was recently

shown that the ability to activate vitamin D is impaired in CF(25), and this may explain the

infection susceptibility and pro-inflammatory shift in CF. Moreover, it would indicate that giving

CF patients active vitamin D may be better than administering the inactive form.

Furthermore, this is the first study that examines the correlation between serum vitamin D

levels in CF patients and the amount of days in hospitalization of all causes. DOHA represents a

general health index in CF, and it is found to be inversely correlated to serum vitamin D levels.

However, there was no significant decrease in DOHA during the follow-up period. This can be

explained either by insufficient rise in serum vitamin D levels during this trial or by the right-

skewed distribution and the high standard deviation of the DOHA parameter, that requires a

bigger sample size in order to show significant difference.

This description of serum 25(OH)D levels being correlated to a general health index is

supported by a meta-analysis of randomized controlled trials which concluded that supplementing

any patient with vitamin D decreased general mortality rate(26). Specifically regarding

respiratory health, vitamin D deficiency increased the risk for upper respiratory tract infections

and decreased FEV1 in asthma and other wheezing diseases(27) in otherwise healthy children. In

order to explain these findings we need to explore the role of vitamin D in the systemic immune

system at first, and then in the respiratory system in particular.

The discovery of VDR and hydroxylase enzymes in immune cells led to a surge of

research on the potential role of vitamin D in maintaining immune homeostasis and preventing

development of autoimmune processes. Vitamin D can directly inhibit antigen presenting cells

by inhibiting expression of major histocompatibility complex (MHC) class II and monocytes

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differentiation to dendritic cells(23). Additionally, it inhibits expression of inflammatory

cytokines (such as IL-1α, IL-1β, TNF-α), and its inhibition on releasing IL-12 from dendritic

cells effects prominently on differentiation of T lymphocytes(28). Vitamin D functions similarly

in the respiratory epithelial cells, and specifically in CF it inhibits secretion of IL-6 and IL-8 after

exposure to antigen and increases levels of human cathelicidin antimicrobial peptide (hCAP18)

mRNA and its protein product(10,29). These findings suggest that vitamin D can enhance

antimicrobial activity in the respiratory epithelium in CF, and may provide a novel therapy for

prevention and treatment of airways infections in this disorder.

Another mechanism in which vitamin D can be helpful in CF is protection against viral

infections. Viral infections, such as respiratory syncytial virus (RSV), influenza A, influenza B

and rhinovirus are a substantial cause of respiratory exacerbations in children with CF and

account for 60% of exacerbations(30). Early epidemiologic studies found a strong association

between rickets and respiratory tract infections(31), and a recent large cross-sectional study

reported that vitamin D status is inversely correlated to upper respiratory tract infections. This

association can be even stronger in patients with chronic respiratory disorders, like CF(32).

Schögler et al.(33)reported recently that vitamin D has a repressive effect on rhinovirus

replication in primary bronchial epithelial cells of CF and healthy children, possibly associated

with the induction of the antimicrobial peptide LL-37.

However, this study is not free from limitations. As already mentioned, the percentage of

patients who reported poor compliance was very high and might have caused suboptimal results.

In addition, this study lasted for only 2 years, while some of the patients were followed for only a

year, and it might be too short a period to show a significant difference. Lastly, other

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confounding factors that are known to decrease pulmonary exacerbations such as vitamin A and

vitamin E were not tested, and this may affect the results.

5. CONCLUSION

Our study demonstrates that the new North American CF Foundation guidelines for

supplementing vitamin D in CF patients succeed in raising serum levels of 25(OH)D, but are still

not sufficient in raising it in all the patients above the cutoff of 30 ng/ml (75 nmol/L). Monitoring

serum vitamin D levels while adjusting the dose according to the levels may lead to normalization

of the levels in most patients. More research is needed to test the protocol over longer periods

with a larger sample size and higher compliance rate in order to draw reliable conclusions.

However, this study suggests that increasing serum vitamin D levels in CF patients leads to a

decrease in the number respiratory exacerbations.

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Geiser T, Regamey N, Alves MP. Vitamin D represses rhinovirus replication in cystic
fibrosis cells by inducing LL-37. Eur. Respir. J. 2016;47(2):520–30.

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FIGURE LEGENDS

Figure 1A – the correlation between the change in vitamin D (in ng/ml) and the change

in DOHA. The figure demonstrates an inverse association (correlation coefficient = -0.484,

p<0.001). DOHA – Days of hospitalization, average number per year per patient. Vitamin D

CHANGE is calculated by the difference between vitamin D levels at the end of follow-up and

vitamin D levels at baseline, so a positive value suggests a net increase in vitamin D levels.

DOHA CHANGE is calculated by the difference between DOHA at the end of follow-up and

DOHA at baseline, so a positive value suggests a net increase in DOHA.

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Figure 1B – the correlation between the change in vitamin D (in ng/ml) and the change in PEA.

The figure demonstrates an inverse association (correlation coefficient = -0.318, p=0.002). PEA

– Pulmonary exacerbations, average number per year per patient. PEA CHANGE is calculated

by the difference between PEA at the end of follow-up and PEA at baseline, so a positive value

suggests a net increase in PEA.

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 Table 1 – Permanent Baseline Characteristics
Variables Absolute Relative

Age (years)
1-10 27 30.0
10-18 22 24.4
+18 41 45.6
Sex
Male 49 54.5
Female 41 45.6
CFTR Mutation
ΔF508/ΔF508 7 7.8
W1282X/W1282X 14 15.6
ΔF508/W1282X 16 17.8
ΔF508/other 14 15.6
W1282X/other 20 22.2
Other 15 16.7
Unknown 4 4.4
a,b
Pancreatic status
PI 74 82.2
PS 16 17.8
Compliance c
GC 31 34.4
PC 59 65.6

Table 1 – the table outlines the subjects distribution for variables registered at baseline and are

not changeable during follow-up.

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a
Exocrine pancreatic function.
b
PI – Pancreatic insufficiency; PS – Pancreatic sufficiency
c
GC – Good compliance; PC – Poor compliance

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 Table 2 – Variable Baseline Characteristics Compared to End of

Follow-up
Variables Baseline End of p Value

BMI
Average 19.40 19.74
Median 18.66 19.13
SD 3.87 3.83

25(OH)D [ng/ml (nmol/L)]

Average 20.97 (52.34) 25.41 (63.42) <0.001
Median 19.50 (48.57) 24.50 (61.15)
SD 11.29 (28.18) 10.73 (26.78)
Calcium (mmol/L)
Average 2.36 2.38 0.2
Median 2.37 2.38
SD 0.11 0.13
Phosphorus (mmol/L)
Average 1.28 1.31 0.2
Median 1.30 1.31
SD 0.30 0.29
PFTs (% of predicted) d
FEV1 76.46 76.83 0.7
FVC 84.00 85.81 0.1
FEV1/FVC 92.58 90.32 0.002

DOHA (days) e
Average 20.00 19.04 0.26
Median 5.00 0.5
SD 33.02 40.48
PEA (number)f
Average 2.79 2.15 0.007
Median 1.50 1.50
SD 3.96 2.91
Vitamin D groups
Vit D<20 45 24
20 ൑VitD < 30 25 39
Vit D ≥ 30 20 27

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Table 2 – the table outlines the mean and median values calculated for every variable measured

during the follow-up period, and compares between baseline and end-of-follow-up values.
d
The values mentioned are average values only. PFTs – Pulmonary function tests; FEV1 –

Forced expiratory volume in 1st second; FVC – Forced vital capacity.

e
DOHA – average number of days of hospitalization (per year per patient).

f
PEA – average number of pulmonary exacerbations (per year per patient).

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 Table 3 – Correlations Between Vitamin D and Other Variables

Variables Pearson Correlation P Value

Coefficient
Calcium 0.027 0.800
Phosphorus -0.295 0.005
g
PFTs
FEV1 0.066 0.559
FVC 0.144 0.198
FEV1/FVC -0.130 0.249
DOHA h -0.484 <0.001
PEA i -0.318 0.002

Table 3 – the table outlines the correlation coefficients between the change in 25(OH)D levels

and the other variables measured, by calculating Pearson's correlation coefficient.

g
PFTs – Pulmonary function tests; FEV1 – Forced expiratory volume in 1st second; FVC –

Forced vital capacity.

h
DOHA – average number of days of hospitalization (per year per patient).

i
PEA – average number of pulmonary exacerbations (per year per patient).

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