Effects of Vitamin D3 Supplementation on Lung Functions and Exercise Tolerance in D3 Deficient Asthma COPD Overlap (ACO) Patients
Phase: PHASE2
Conditions: Asthma-COPD Overlap Syndrome
Sponsor: Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh

 
 
1 
 
                  
 
 
 
 
                      RESEARCH PROTOCOL 
 
 
 
 
 
TITLE 
Effects of vitamin D3 supplementation on lung functions and 
exercise tolerance in D3 deficient Asthma COPD Overlap 
(ACO) patients 
 
 
                                           Date:06/09/2018 
 
 
 
 
 

 
 
2 
 
 
 
Project Summary  
Background: Asthma COPD Overlap (ACO) has been described as a 
distinct disease now-a-days. Several studies have shown positive 
relationship between serum concentration of 25 hydroxyvitamin D, lung 
function and exercise tolerance in different group of patients. However, 
vitamin D   supplementation showed significant improvement in lung 
function status and exercise tolerance in both asthma and COPD patients 
but no study was found in ACO patients. Objectives: To evaluate the 
effects of vitamin D3 supplementation on lung function status and 
exercise tolerance in D3 deficient, stable patients with ACO. Methods: 
This double blinded placebo controlled trial will be conducted by 
Department of Physiology Bangabandhu Sheikh Mujib Medical 
University (BSMMU), Shahbag, Dhaka from March 2018 to February 
2019. For this study a total number 70  vitamin D3 deficient (serum 25 
hydroxycholecalceferol <30 ng/ml) male, stable (diagnosed patient, who 
was not experienced any acute exacerbation, hospitalization, urgent care 
visits or changes in routine medication within 4 weeks prior to study) 
patients with ACO of age 40-70 years will be selected. They will be 
selected from the Out Patient Department (OPD) of the National 
Institute of Diseases of Chest and Hospital (NIDCH) and will be 
randomly grouped as A (control) and B (study). All the participants will 
be matched in terms of age, duration of ACO , duration of smoking, 
body mass index (BMI), mid upper arm circumference (MUAC), waist 
hip 
ratio, 
occupational 
status, 
socioeconomic 
status, 
serum 

 
 
3 
 
25hydroxycholecalceferol [25(OH)D], serum parathormone (PTH) , 
serum calcium (Ca2+), serum phosphate ( PO4
3-) ,serum alkaline 
phosphatase (ALP), serum glutamate pyruvate transaminase (SGPT), 
serum creatinine, fasting blood sugar (FBS), serum glycated 
haemoglobin (HbA1C), serum cholesterol, serum high density 
lipoprotein (HDL), serum low density lipoprotein (LDL), serum 
triglyceride (TG). Any patient with history of any acute exacerbation of 
any pulmonary, liver, endocrine or renal disease, or with any malignancy 
or with any drug known to affect vitamin D metabolism within 1 month 
prior to study, will be excluded from the study. All the baseline data at 
day 0 will be recorded as A0 (Control) and B0 (Study). Along with the 
standard pharmacological treatment of ACO (according to GOLD 
criteria), the patients of the ‘Study group’ will be prescribed for 80,000 
IU of oral vitamin D3 per week and ‘Control group’ for placebo for 
consecutive 13 weeks. Subsequently, all the patients will be cordially 
requested to again visit the Department of physiology, BSMMU, on 13th 
week of their follow up to reexamine all the spirometric variables along 
with serum 25(OH)D and Ca2+ (to check the toxicity). These data will be 
recorded as values of ‘13th week’ (B1). Then according to serum level of 
25(OH)D and  Ca2+ (American Vita min D council 2017), 40,000 IU / 1 
to 6 week oral vitamin D3 will be prescribed, for further 13 weeks. At 
26th week of follow up, all the study variables will be again reexamined, 
and the data will be recorded as values of ‘26th week’ (B2). Similarly, all 
the patients of ‘control group’ will be prescribed for placebo (along with 
standard 
pharmacological 
treatment 
of 
ACO, 
prescribed 
by 
pulmonologist) for consecutive 26 weeks and their respective data will 
be recorded as A1 and A2. With this, all patients of both the groups were 

 
 
4 
 
advised to continue ad lib (according to their own choice) diet. At the 
very 1st day of the study, the spirometric lung functions will be assessed 
by measuring forced vital capacity (FVC), forced expiratory volume in 
1st second (FEV1), forced expiratory ratio (FEV1/FVC%), peak 
expiratory flow rate (PEFR) and forced mid expiratory flow of FVC 
(FEF25-75%), maximum expiratory flow75 (MEF75), maximum expiratory 
flow50 (MEF50), maximum expiratory flow25 (MEF25) with a portable 
digital spirometer with a portable digital spirometer. In addition, exercise 
tolerance will be assessed by change in 6 minute walk distance (6MWD) 
in 6 minute walk test (6MWT). Changes in peripheral capillary oxygen 
saturation (SpO2) by Pulse Oximeter and degree of dyspnea and fatigue 
by Modified Borg Scale (MBS) will also be measured both before and 
after 6MWT, to evaluate their changes in both the groups. All these 
variables will be again measured on their follow-up in 13 weeks and 26 
weeks with standard pharmacological treatment of ACO with D3 
intervention (B group) and also without D3 intervention (A group). The 
data will be statistically analyzed by Graphpad prism (Version 7) using 
independent sample ‘t’ test (between two groups), paired Student’s ‘t’ 
test (between paired groups before and after intervention). Group 
comparisons will be done using one way ANOVA.  In the interpretation 
of results, ≤0.05 level of probability (p) will be accepted as significant.                         
Expected outcome of the study: 
 Impaired lung functions  and exercise tolerance in vitamin 
D3  deficient  ACO patients will be detected and the good 
impact of vitamin D3 supplementation for six months on 
restoration of  the lung function status and exercise 
tolerance in ACO patients  will be proved. 

 
 
5 
 
Key words: Asthma COPD Overlap, Vitamin D3, FVC, FEV1, 
FEV1/FVC%, FEF25-75%, PEFR, MEF75% , MEF50%, MEF25%, SpO2, 6 
Minute Walk Distance, Modified BORG scale. 
Introduction                                                                                                                
Chronic obstructive pulmonary disease (COPD) and bronchial asthma 
both are common airway diseases contributing to mortality and 
morbidity worldwide. There is growing consensus that typical asthma 
and COPD characteristics can both exist simultaneously in one patient. 
Approximately one in four patients with COPD has asthmatic features 
and patients with asthma may present with fixed airway obstruction over 
time. A joint project of Global Initiative for Asthma (GINA) and Global 
Initiative for Chronic Obstructive Lung Disease (GOLD) has 
recommended the term “asthma COPD overlap (ACO)” for these 
patients. ACO is defined as persistent airflow limitation with several 
features associated with asthma and several features associated with 
COPD. ACO is associated with increased disease severity and worse 
health status and poorest health related quality of life. These patients 
experience greater exacerbation  than those with COPD alone, have high 
hospital burden, and require a large amount of medical resources 
compared to patients with asthma or COPD. 
 
Though all of airway diseases (ACO/ COPD/ Asthma) are preventable 
and treatable, once developed the disease along with its comorbidities 
cannot be cured. But their progression and exacerbation of morbidity can 
be reduced. Medicine treatment alone cannot do this. Now-a-days the 

 
 
6 
 
interest of role of different non-pharmacological supplements on 
different lung diseases are outgoing. 
Vitamin D is now known to be of physiological importance outside of 
bone health and calcium homeostasis. There is a large amount of 
evidence supporting the influence of vitamin D on immune function and 
inflammatory diseases. In addition it plays beneficial role in the 
prevention of cancer, autoimmune disease, cardiovascular disorders, 
hypertension and influenza. 
In respiratory health, higher serum vitamin D concentration has been 
associated with better lung function as measured by FEV1 in a large 
cross-sectional study of the US population. Recently a group of 
researcher found a statistically significant positive relationship between 
FEV1 and serum 25(OH)D in young COPD patients. On the other hand, 
statistically significant association between vitamin D deficiency and 
asthma has been reported and this vitamin’s deficiency is associated with 
severe asthma exacerbation and need for emergency department 
evaluation or hospitalization. High vitamin D levels are associated with 
better lung function, less airway hyperresponsiveness and improved 
glucocorticoid response in adult asthma. Although the precise 
connection between vitamin D status and lung function is not clear at 
this point, it is postulated that vitamin D may improve lung function 
through its action on regulating inflammation inducing antimicrobial 
peptides, and/or its action on muscles. 

 
 
7 
 
Plasma vitamin D concentration has been positively associated with 
exercise capacity in COPD patients and it was observed that exercise 
capacity was decreased in vitamin D deficient COPD patients.  
In patients with bronchial asthma significant improvement in FEV1 was 
shown after 50,000 IU /day of  vitamin D for consecuetive 3 months. In 
addition, in D3  deficient COPD patients 50,000 IU/week vitamin D for 8 
weeks followed by 800 IU /day for 1 year did significant improvement 
in exercise tolerance with decrement of frequency of COPD 
exacerbations.In a similar group of patients, only 50,000 IU / week 
vitamin D  for 3 months showed significant improvement in exercise 
tolerance.  
However, as far as it has been reviewed, association of vitamin D status 
with ACO has only been observed by Odler et al. (2015). In that study 
60% of ACO patients had vitamin D deficiency and a positive 
correlation was found between serum  25(OH)D  and spirometric lung 
function status. Strikingly no study have been conducted to observe the 
effects of  this fat soluble vitamin on lung function and exercise capacity  
in vitamin D3 deficient, stable patients with ACO.  
Therefore, on the basis of this background the present study has been 
designed to evaluate the effects of Vitamin D3 on the spirometric lung 
function status, exercise tolerance in D3 deficient, stable patients with 
ACO. This study may draw attention of the physicians about the 
beneficial effects of the vitamin D3 on both pulmonary and 
extrapulmonary menifestation in ACO patients.  
 

 
 
8 
 
Rationale 
In recent years, ACO has gained much attention and have extensively 
reviewed worldwide. There is a scarcity of population-based data on 
ACO in Bangladesh except Habib et al. As patients with ACO seem to 
be at risk for a poor outcome, high risk of exacerbations, it is of utmost 
importance to perform more research on this significant disease. 
 
However, as far as it has been searched association of vitamin D status 
with ACO has only seen by Odler et al. (2015) who reported significant 
decrement of serum 25(OH)D in elderly ACO patients. Strikingly no 
study have been conducted to observe the effects of  this fat soluble 
vitamin on lung function and exercise capacity  in vitamin D3 deficient, 
stable patients with ACO.  
 
To the best of our knowledge, as far as we reviewed with a loading dose 
followed by upto 50,000 IU/week of oral D3 for 12 weeks, could not 
improve the spirometric variables in stable COPD patients (Said and 
Elnaeem 2014; Hornikx et al. 2015; Moosavi and Shoushtari 2015;  
Rezk et al. 2015). Whereas, Nazarian et al. (2011) found significant 
improvement of insulin sensitivity in patients with Type II Diabetes 
meliitus after 4 weeks oral supplementation of 10,000IU/day vitamin D3 
(Nazarian et al. 2011). On the basis of this observation, we have selected 
80,000 IU/ week of oral vitamin D3 , for consecutive 13 weeks followed 
by  40,000 IU / 1 to 6 week for further consecutive 13 weeks, as the 
dosage schedule for our D3 deficient patients of stable ACO. In addition, 

 
 
9 
 
as toxicity dose of vitamin D3 is very high (50,000IU/day for several 
months) (Veith 1999), this dose schedule will be safe for our patients. 
 
Therefore, on the basis of this background the present study has been 
designed to evaluate the effects of Vitamin D3 on the spirometric lung 
function status, exercise tolerance in D3 deficient, stable patients with 
ACO. This study may draw attention of the physicians about the 
beneficial effects of the vitamin D3 on both pulmonary and 
extrapulmonary menifestation in ACO patients.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
10 
 
Research question 
Is there any improvement in lung function status and exercise tolerance 
in vitamin D3 deficient ACO patients after having vitamin D3 
supplementation? 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
11 
 
Hypothesis 
Null: Vitamin D3 supplementation does not improve lung functions and 
exercise tolerance in vitamin D deficient ACO patient. 
 
Alternate: Vitamin D3 supplementation improves lung functions and 
exercise tolerance in vitamin D deficient ACO patient. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Objectives: 
General objectives: 
To evaluate the effects of vitamin D3 supplementation on lung function 
status and exercise tolerance in D3 deficient, stable patients with ACO. 
 
Specific objectives 
 To measure the FVC, FEV1, FEV6, FEV1/FVC%, PEFR and FEF25-
75% of vitamin D3 deficient,  stable patients with ACO, in order to 
assess their lung function status at baseline. 
 To measure the 6MWD of all the patients, in order to assess their 
functional exercise capacity at baseline. 
 To measure the SpO2 at rest of all the patients, in order to assess the 
basal oxygenation status at baseline. 
 To measure the level of dyspnea and fatigue at rest of all the patients 
in order to assess the basal breathlessness level at baseline. 
 To measure the both the SpO2 and level of dyspnea and fatigue again 
after 6MWT, for the assessment of their exercise tolerance at 
baseline. 
 To measure the all these variables in study group at 13th weeks and at 
26th weeks of their follow up.  
 To measure the all these variables in control group at 13th weeks and 
at 26th weeks of their follow up. 
 To compare all these variables in study group at baseline and endline.  
 To compare all these variables in control group at baseline and 
endline  
 To compare all these variables in both group at baseline and endline. 

 
 
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Methods 
For the assessment of all the variables of all the patients on day 0, at 13th 
week and as well as at 26th week, they will be designated as A0, A1 and 
A2 (control) and B0, B1 and B2 (study). In addition, for the mean of 
SpO2, level of dyspnea and level of fatigue at resting condition (before 
6MWT), all the groups will be further  designated as  A0before, A1before, 
A2before, B0before, B1before and B2before respectively. Similarly after 6MWT 
all these six subgroups will be again designated as A0after, A1after, A2after 
and B0after, B1after, B2after, for measurement of these three variables. 
 
Type of study                 : Double blinded placebo controlled trial 
Place of study 
: Department of Physiology, Bangabandhu 
Sheikh Mujib Medical University (BSMMU), 
Shahbagh, Dhaka 
Period of study 
: March 2018 to February 2019 
Study population 
: Stable patients of ACO with vitamin D3 
deficiency 
Sample size 
: 60 
Sampling 
: Consecutive sampling 
Grouping 
:  
Group A (Control) 
: 30 (thirty) D3 deficient ACO patients with 
placebo administration 
Group B (Study) 
: 30 (thirty) D3 deficient ACO patients with 
vitamin D3 administration           
 

 
 
14 
 
 
Sample size calculation 
Using the following statistical formula 
n =    [(Zα+Zβ)2 X (σ12+σ22)] / (μ1-μ2)2   
                                                                                                              (Kirkwood and Sterne 2003)
 
` 
Therefore, 
 n  
=    [(Zα+Zβ)2 X (σ12+σ22)] / (μ1-μ2)2 
=    [(1.96+0.84)2 X (7.212 +12.252)] / (364.1-357.3)2 
=    [7.84 X (51.9841+150.0625)] / (6.8) 2 
=    1584.04534/ 46.24 
=    34.2570359 
          =    approximately 35 for each group. 
 
 
Mean of control,  
 
μ1 
= 
364.1 
Mean of study group,  
 
μ2 
= 
357.3 
SD of control,  
σ1 
= 
7.21                                        
SD of study group,  
 
σ2 
=  
12.25                                             
 
 
 
                         (Hassan 2018) 
Type I Error,      
           
Zα 
= 
1.96 
Type II Error,              
Zβ   
= 
0.84 
 
 
 
 
      (Kirkwood and Sterne 2003) 

 
 
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Subject selection: 
Inclusion criteria 
  Pulmonologist diagnosed stable patient of ACO 
(GINA and GOLD 2015) 
   Duration of ACO: > 4 year 
 
   Vitamin D3 deficient : Serum 25(OH)D <30 ng/ml 
(Vitamin D Council 2017) 
   Age: 40 -70 years 
   Sex: Male 
   Socioeconomic status: middle class 
   Smoker 
   Anthropometric status:  
o BMI=18.6-24.9 kg/m2 
(WHO 2006)  
o mid upper arm circumference>25.1 cm  
(Sultana et al. 2015) 
o waist-hip ratio<0.89 
(Sultana et al. 2015) 
   Serum parathormone: 10-65 pg/ml 
   Serum Ca2+: 8.5-10.5 mg/dl 
   Serum PO4
3: 2.3-4.7 mg/dl 
   Serum alkaline phosphatase: 30-120 U/L 
   SGPT: <50 U/L 
   Serum creatinine: 0.7-1.3 mg/dl  
   FBS: 3.5-6.1 mmol/L  
   Serum HbA1c: 4.5-6.3 %  

 
 
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   Serum cholesterol: <200 mg/dl 
   Serum HDL: >40 mg/dl,  
   Serum LDL: <130 mg/dl 
 Serum TG: <150 mg/dl 
Exclusion criteria  
     With history of:  
 any other pulmonary diseases, as, 
 chronic obstructive pulmonary disease 
 bronchial asthma 
 respiratory tract infection 
 bronchiectasis       
 pleural effusion  
 tuberculosis           
 interstitial lung disease  
 pneumonectomy or pulmonary lobectomy 
 any cardiac disease, like –  
 unstable angina pectoris 
 congestive heart failure       
 myocardial infarction 
 cardiac arrhythmia 
 systemic hypertension  
 any liver disease 
 any malignancy  

 
 
17 
 
 use of any drugs known to affect vitamin D metabolism 
within 1 month prior to study, as, 
 antiepileptics (Phenytoin, Carbamazepine) 
 antibiotics (Clotrimazole, Rifampicin) 
 antihypertensives (Nifedipine, Spironolactone) 
 antiretroviral drugs (Ritononavir, Saquinavir) 
 endocrine drugs (Cyproterone acetate) 
 glucocorticoids 
 bisphosphonate 
 calcium supplement 
 With biochemical evidence of 
 diabetis mellitus 
 renal insufficiency 
All the criteria mentioned above will be scrutinized by taking history or 
clinical examination or laboratory investigations 
 
 
 
 
 
 
 
 

 
 
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Study outline: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Selection of diagnosed ACO patients 
According to inclusion and exclusion criteria 
Final enrollment of 60 samples 
Randomization 
GroupA:Control group 
        n= 30 
Group B: Study group 
              n= 30 
Group A0: Baseline 
data recording at day 
‘0’ 
Group A1: Follow up 
data 
recording 
at 
‘13’week. 
Group A2: Follow up 
data 
recording 
at  
‘26’week 
 
Group B0: Baseline 
data recording at day 
‘0’ 
Group B1: Follow up 
data 
recording 
at 
‘13’week 
Consecutive sampling 
Group B2: Follow up 
data 
recording 
at 
‘26’week 

 
 
19 
 
 
 Site of sample collection 
All the patients will be collected from the Out Patient Department (OPD) 
of National institute of the diseases of chest and hospital (NIDCH), 
Mohakhali, Dhaka.. 
 
Study procedure   
On the first day of enrollment, the objectives, nature, purpose and 
potential risk of all the procedures used for the study will be explained in 
detail to each ACO patients (diagnosed by pulmonologist), with a cordial 
attitude giving emphasis on the benefits he might obtain from this study. 
He will be encouraged for voluntary participation and will be allowed to 
withdraw himself from the study even after participation, whenever he 
felt uneasy. If he agreed to be enrolled in the study, an informed written 
consent will be taken in a prescribed form. Detailed family history, 
medical history and thorough physical examination of each patient will 
be done and all the information will be recorded in a standard data sheet. 
Then all the patients will be requested to attend the Department of 
Physiology at 7:30 am (after overnight fasting) on the examination day.  
 
On the examination  day, 10 ml of venous blood will be  collected from 
antecubital vein of  patient in different vaccutainer tubes and will be 
taken to the laboratory of Department of Biochemistry and Molecular 
Biology . as soon as possible for the estimation of  serum creatinine and 
serum 25(OH)D. and rest of the blood will be preserved at -4˚C.  
 

 
 
20 
 
If the patient  with D3 deficiency [Serum 25(OH)D <30 ng/ml]  and not 
with renal insufficiency (Serum creatinine >1.3 mg/dl) , then the Fasting 
blood sugar, serum HbA1C, serum parathormone (PTH), serum calcium 
(Ca2+), serum phosphate (PO4
3-), serum alkaline phosphatase (ALP), 
serum SGPT, serum cholesterol, serum High Density Lipoprotein 
(HDL), serum low density lipoprotein (LDL), serum triglyceride (TG),  
will be assessed from the preserved blood.          
 
After that their height and weight will be measured and the spirometric 
lung function test will be done by using a portable spirometer (PONY 
FX, cosmed, Italy]. After getting all the biochemical and spirometric 
reports the final selection will be done, according to the inclusion and 
exclusion criteria. All the eligible patients will be randomly assigned to 
either ‘Control’ or ‘Study’ groups and will be thoroughly informed about 
the objectives and detailed study procedure, once again. 
Subsequently a standard therapeutic treatment (according to GOLD 
guideline) will be prescribed to all the selected stable ACO patients of 
both groups. Proper education will be given about drug, method of 
taking medication and  medication  plan. 
 
Along with the standard pharmacological treatment of ACO, the patients 
of the ‘Study group’ will be prescribed for 80,000 IU/ week of oral 
vitamin D3 , for consecutive 13 weeks. Subsequently, all the patients will 
be cordially requested to again visit the Department of physiology, 
BSMMU, on 13th week of their follow up to reexamine all the study 

 
 
21 
 
variables along with serum 25(OH)D and Ca2+ (to check the toxicity). 
These data will be recorded as values of ‘13th week’ (B13). 
 
Then according to serum level of 25(OH)D and  Ca2+ (American 
Vitamin D council 2017), 40,000 IU / 1 to 6 week oral vitamin D3 will 
be prescribed, for further 13 weeks. At 26th week of follow up, they will 
be requested again to visit the Department of Physiology, BSMMU, to 
reexamine all the study variables, and the data will be recorded as values 
of ‘26th week’ (B26).  
 
Similarly, all the patients of ‘control group’ will be prescribed for 
placebo (along with standard pharmacological treatment of ACO, 
prescribed by pulmonologist) for consecutive 26 weeks and their 
respective data will be recorded as A13 and A26.  
 
With these, all patients of both the groups will be advised to continue ad 
lib (according to their own choice) diet as well as to have sunlight 
exposure (within 10 am to 2 pm) only for 20 minutes daily (Manjeeta 
2016).  
 
In the mean time, a good rapport will be built up to take time to time 
follow-up over telephone and visiting patient’s place with scheduled 
appointment  to maintain a proper follow up at 1st (day 1) 2nd (at 13th 
week) and 3rd (at 26th week) visit of the study , in addition to the 
hematological values. 
 

 
 
22 
 
Any patient, who will be failed to follow the study procedure exactly 
during study period, will be dropped and a new one will be included to 
fulfill the desired total sample number. 
 
 Test procedure for spirometry  
                                                                                          (Hassan 2018) 
 At first, the subject will be asked to take rest sitting on a chair and 
remain calm and quiet for 5 minutes. 
 The detailed procedure will be explained to him to ensure his 
maximum cooperation. 
 The switch of spirometer will be on and windows will be open. 
 Patients’ information will be filled up and saved. 
 The patient will be asked to hold a disposable paper mouthpiece 
(connected to spirometer) in his hand horizontally and to place it in 
between his two lips. 
 To make good seal, he will also be asked to put the lips tightly 
around the outside of the mouthpiece. 
 Then he will be asked to inhale first as deeply and as rapidly, as 
possible, which was followed by a forceful expiration for the 
possible longest period into the mouthpiece. 
 The readings of different variables will be recorded from the 
spirometer monitor. 
 Three (3) consecutive readings at 5 minutes interval for each 
parameter will be taken and the best value will be noted. 
  

 
 
23 
 
Test procedure for pulse oximetry  
                                                                                         (Hassan  2018) 
 The patient will be asked to take rest and remain calm and quiet for 
10 minutes (MacAllister et al. 2007).  
 The detailed procedure will be explained to him to ensure his 
maximum cooperation. 
 Then his right index finger will be washed with alcohol and pulse 
oximeter sensor will be applied.  
 The switch button will be on. 
 The reading will be recorded from the display screen. 
 Three (3) consecutive readings at 5 minutes interval will be taken 
and the best value will be noted. 
 
Procedure for six minute walk test (6MWT)  
                                                                                  (Hassan  2018) 
The 6MWT will be performed at indoors, along a long, flat, straight, 
enclosed corridor with a hard surface that was seldom traveled. The 
walking course will be 30 meter in length which was marked every 3 
meter. The turnaround points will be marked with a cone. A starting line, 
which marked the beginning and end of each 6 meter lap, will be marked 
on the floor using brightly colored tape. 
 
The subject will be asked to walk at his own maximal pace along the 
corridor from the end, covering as much ground as he can, during the 
allotted time, without running. He will be also advised to take rest if he 

 
 
24 
 
was too exhausted to continue the test. Standard phrases at regular 
intervals (every 60 seconds) will be used, like: “you are doing well,” 
“keep up the good work”. At the same time he will be also informed 
about the past time and how much left before the test completed.  
 
If any of the subjects develop chest pain, intolerable dyspnea, leg 
cramps, staggering, diaphoresis or pale appearance the test will be 
immediately stopped and the subject will be dropped. Again, when any 
subject will complete the total test procedure (according to the exact 
protocol), he will be congratulated for good effort. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
25 
 
Study variables   
 
   Units           
 Lung function variables 
 
 
 Forced Vital Capacity (FVC)  
Liters 
 Forced Expiratory Volume in 
1st second (FEV1) 
 
Liters 
 FEV1 /FVC ratio  
 
% 
 Peak expiratory flow rate  
(PEFR) 
 
liters/s 
 Forced Expiratory Flow in 
the middle half of FVC 
(FEF25-75) 
 
liters/s 
 Maximum Expiratory Flow 
at     25% of the FVC 
(MEF75) 
 
liters/s 
 Maximum Expiratory Flow 
at 50% of the FVC (MEF50) 
 
liters/s 
 Maximum Expiratory Flow 
at 75% of the FVC (MEF25) 
 
liters/s 
 Exercise tolerance variables 
 
 
  Six Minute Walk Distance 
(6MWD)       
 
Meters 
 Peripheral Capillary Oxygen  
saturation (SpO2) 
 
% 
 Level of Dyspnea 
 
     Borg score 
 Level of Fatigue 
 
     Borg score 

 
 
26 
 
 
 
 
Vitamin and placebo administration 
 
 
Vitamin D3 
 
Placebo 
Collection Beximco Pharmaceuticals 
Limited, Bangladesh 
 
Beximco 
Pharmaceuticals 
Limited, Bangladesh 
Dose 
80,000 IU/wk   
 
80,000 IU/wk   
Route 
Oral    
 
Oral    
Ingredient 
 Cholecalciferol 
(40,000IU) 
 Microcrystalline 
 Cellulose (58.1 
gm) 
 Butylated Hydroxy 
Toluene ( 0.2mg) 
 Magnesium Stearate 
(3mg) 
 Gelatin Capsule 
Shell  (1mg) 
 
 
 
 Microcrystalline 
 Cellulose (303.8gm) 
 Butylated Hydroxy 
Toluene (0.2mg) 
 Magnesium Stearate 
(3mg) 
 Gelatin Capsule Shell  
(1mg) 
 
 
 

 
 
27 
 
            
 
 
 
 
 
 
 
                  STATISTICAL ANALYSIS 
 
 
 
TITLE 
Effects of vitamin D3 supplementation on lung functions and 
exercise tolerance in D3 deficient Asthma COPD Overlap 
(ACO) patients 
 
 
                                        Date: 06/09/2018 
 
 
 
 
 

 
 
28 
 
 
 
 
 
The results will be expressed as mean with standard error  (mean±SE). 
The data will be statistically analyzed by Graphpad prism (Version 7) 
using independent sample ‘t’ test (between two groups), paired Student’s 
‘t’ test (between paired groups before and after intervention). Group 
comparisons will be done using one way ANOVA. Correlations between 
variables will be tested with Pearson correlation coefficient. 
 
In the interpretation of results, ≤0.05 level of probability (p) will be 
accepted as significant. 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
29 
 
 
 
. 
   
 
 
 
           INFORMED CONSENT STATEMENT 
 
 
TITLE 
Effects of vitamin D3 supplementation on lung functions and 
exercise tolerance in D3 deficient Asthma COPD Overlap 
(ACO) patients 
 
 
 
                                      Date :06/09/2018 
 
 
 
 
 
 

 
 
30 
 
 
Title of the study: Effects of Vitamin D3  supplementation  on  lung functions 
and exercise tolerance in D3 deficient Asthma COPD Overlap  (ACO) patients.                            
Researcher:  
Dr. Naznin Sultana 
MD Resident 
Department of Physiology 
Bangabandhu Sheikh Mujib Medical University (BSMMU) 
Shahbag, Dhaka, Bangladesh. 
 
Supervisor: 
Dr.Taskina Ali 
Associate Professor 
Department of Physiology 
Bangabandhu Sheikh Mujib Medical University (BSMMU),  
Shahbag, Dhaka, Bangladesh.  
 
You are being asked to take part in a research study. Your participation is voluntary. 
Before agreeing to participate in this study, it is important that you read the 
following explanation of the study, if you do not understand what you are reading, 
do not sign it. Please ask the researcher to explain anything, you do not understand. 
If you decide to participate, you will be asked to sign this form and a copy will be 
given to you. 
Purpose of the study: The present study will be conducted to assess                         
lung function status and exercise tolerance in D3 deficient ACO patients                         
before and after taking vitamin D therapy for a period of 6 months. This study may 
find out the benefit of vitamin D therapy on lung function and exercise tolerance  in 
ACO patients. 
Explanation of procedures: The researcher will take an interview and will do 
general examination of the body. Then the spirometric variable will be measured  
and exercise tolerance, oxygen saturation, the level of dyspnea and fatigue (Borg 

 
 
31 
 
scale)  will be measured before and after six minute walk test . Then 5 ml venous 
blood will be collected under all aseptic precaution. The same procedure will be 
done on his follow up.  
Risks and discomfort: This study involves almost no physical risk. However, while 
drawing the blood sample the subject may experience slight discomfort but it will be 
negligible. Adequate precautions will be taken to avoid any error. 
Withdrawal without prejudice: Each participant is free to withdraw the consent 
and discontinue participation in this study at any time. 
Confidentiality: Your identity in this study will be treated as confidential. Your 
name or identity will not be linked in any way to the research study. 
Costs and payments to subjects for participation in research: There is no 
financial support (compensation) for your participation in this research. 
Available medical treatment for research related injuries: If you are injured as a 
direct result of taking part in this research study, emergency medical care will be 
provided by transporting to your personal doctor or medical centre. 
Questions: Any question concerning the research, study participants can call Dr. 
Naznin Sultana, Mob no-01798598493.    
Authorization: I have read and understood this consent form and I volunteer to 
participate in this research study, but I understand that my consent does not take 
away any legal rights in the case of negligence or other legal fault of anyone who is 
involved in this study. 
 
 
 
Participant’s Signature:........................................ 
Date:.................................. 
 
 
 
 
 

 
 
32 
 
 
Appendix-I 
Clearance from Institutional Review Board (IRB) of BSMMU 
 
 
 

 
 
33 
 
 
 
Appendix-II 
                                 DATA  SCHEDULE                                  
Title : Effects of vitamin D3  supplementation  on  lung functions and exercise 
tolerance in D3 deficient ACO patients . 
SL No. 
:                     
ID No. 
:                     
Date 
:                   
 
Subject information: 
Name  
:   
Age 
:  
NID No 
:  
Contact address 
 
 
: 
 
Permanent 
address 
 
 
: 
 
Contact No 
: Patient:                                
Attendant: 
Monthly income 
(taka) 
 
: 
 
No. 
of 
family 
member 
 
: 
 
Residence 
: Urban/Rural 
Occupation 
: Homemaker-1,Student-2,Retired-3, 
Business-4, Service-5, Day labourer-6 
Education 
: Illiterate-0, Primary-1, High school-2, SSC-3, 
HSC-4, Graduate-5, Masters-6 
 
 
 

 
 
34 
 
Marital status 
: Married-1, Unmarried-2, Widower-3,  
Separated-4,Divorced-5,Livetogether-6 
Biomass 
fuel 
exposure 
: Yes/No 
Smoking habit 
: Current/Past smoker 
: Duration of smocking: 
: Stopped for (in years, if past smoker): 
: Packs-year:                     (sticks per day/20 Χ 
year) 
 
Past medical history: 
 
History of ACO: 
H/O  Hypertension 
: Yes/No     If yes,duration                months               
H/O  Diabetes mellitus 
: Yes/No     If yes,duration                months               
H/O  Cardiac disease 
: Yes/No    If yes, duration                months               
H/O  Renal disorder 
: Yes/No    If yes, duration                months               
H/O Endocrine disorder 
: Yes/No    If yes, duration                months               
H/O  Psychosis 
: Yes/No    If yes, duration                months               
H/O Rheumatoid arthritis 
: Yes/No    If yes, duration                months               
H/O Other disease 
: Yes/No   If yes, duration                 months 
Drug history 
:  
Place of diagnosis 
: 
 
Duration of illness 
: 
 
 

 
 
35 
 
 
 
 
 
Features 
Favors Asthma 
Favors COPD 
Age of onset 
□ Before age 20 years 
□ After age 40years 
Pattern of  
respiratory  
symptoms 
□ Variation in symptoms over 
minutes,hours or days  
□Persistence 
of 
symptoms 
despite 
treatment 
□ Symptoms worse during night or 
early morning 
□ Good or bad days but 
always daily symptoms 
& exertional dyspnea 
□ 
Symptoms 
triggered 
by 
exercise,emotions 
including 
laughter,dust or allergen 
□Chronic 
cough 
& 
sputum 
preceded 
dyspnea, unrelated to 
triggers 
Lung function 
□Record 
variable 
airflow 
limitation(spirometry, peakflow) 
□Record 
persistant 
airflow 
airflow 
limitation(post 
BD  
<0.7) 
Lung 
function  
between 
symptoms 
□Normal 
□Abnormal 
Past 
history 
or 
family history 
□ Previous doctor diagnosis of 
asthma 
□ 
Previous 
doctor 
diagnosis 
of 
COPD,chronic 
bronchitis or 
Emphysema 
□ Family history of asthma and 
otherallergic condition 
□ Heavy exposure to 
tobacco smoke, biomass 
fuel 
Time course 
□ Symptoms vary seasonally, or 
from year to year  
□ 
Symptoms 
slowly 
worsening 
over 
time(progressive) 
□ May improve spontaneously or 
have an immediate response to BD 
or to  ICS over weeks 
□ 
Rapid 
acting 
bronchodialator 
provides only limited 
relief.  
Chest X-ray 
□Normal  
□Severe hyperinflation 

 
 
36 
 
History of sun exposure: 
Time 
: 
             /day or week or month              
Duration 
: 
             /min or hour 
Anthropometric data: 
Height :             cm            Weight:            kg 
 BMI:                    kg/m2         
Waist:                cm 
   Hip:               cm 
 Waist-hip ratio: 
MUAC:              cm 
 
 
General examination: ( present = +, absent = -) 
Anaemia 
- 
/ + / ++ / +++ 
Jaundice 
- 
/ + / ++ / +++ 
Cyanosis 
- 
/ + / ++ / +++ 
Clubbing 
- 
/ + / ++ / +++ 
Edema 
- 
/ + / ++ / +++ 
Ascites 
- 
/ + 
 
 
 
 
Systemic 
examination: 
Respiratory system: 
Shape of the chest 
: 
Normal-1;abnormal-2 
Movement of chest 
: 
Abdominothoracic-1;  
 thoracoabdominal-2 
Tracheal position 
: 
Central-1;shifted to right-2; 
shifted-3 
Use 
of 
accessory 
respiratory muscle 
 
: 
       
Yes-1;no-2 
Percussion note 
: 
Dull-1;resonant-2 
Breath sound 
: 
Vesicular-1;bronchial-2 
Added sound 
: 
No-1;ronchi-2;creps-3 
 Pulse rate 
 (Beats/min) 
: 
 BP 
 (mmHg) 
:  Systolic:                    
   Diastolic: 

 
 
37 
 
Other system: 
Heart 
: 
Not palpable-1;palpable-2 
Kidney 
: 
Not palpable-1;palpable-2 
Spleen 
: 
Not palpable-1;palpable-2 
Liver 
: 
Not palpable-1;palpable-2 
Lung function test: 
Variables 
MV 
Day 1 
(post 
BD) 
% of PV 
Day 1 
MV 
Day 90 
(post 
BD) 
% 
of 
PV 
Day  
90 
MV 
Day 90 
(post 
BD) 
% of PV 
Day 180 
    
 
 
 
 
 
FVC(L) 
 
 
 
 
 
 
FEV1(L) 
 
 
 
 
 
 
FEV1/FVC(L) 
 
 
 
 
 
 
PEFR(L/S) 
 
 
 
 
 
 
FEF 25-75% 
 
 
 
 
 
 
(PV =Predicted Value , MV=Measured Value) \ 
Exercise tolerance variables 
(a) 6MWD  
   (meter) 
On day 
 0 
On day 
 90 
On day  
180 
No. of laps……………(Χ 60 min)+ final 
partial lap………meter 
 
 
 
Stopped or paused before 6min   (yes/no)     
Reason 
     
  
   
Symptoms 
at 
the 
end 
of 
exercise: 
angina/dizziness/hip,leg or cuff pain 
 
 
 
          

 
 
38 
 
Haematological variables: 
 
 
 
 
 
 
 
    On day 
         0 
     On day 
        90 
    On day 
      18 0 
(b)(SpO2)(%) 
Before 
6MWT 
After 
6MWT 
Before 
6MWT 
After 
6MWT 
Before 
6MWT 
After 
6MWT 
(c) 
Level 
of 
dyspnea(score) 
 
 
 
 
 
 
(d) 
Level 
of 
fatigue(score) 
 
 
 
 
 
 
Variables  
On day 
  1 
On day  
90 
On day 180 
Serum 25(OH)D  (mg/ml) 
 
 
 
Serum  Ca2 (mg/dl) 
 
 
 
Serum  PO4
3- (mg/ml) 
 
 
 
Serum  PTH  (pg/ml) 
 
 
 
Serum creatinine(mg/dl) 
 
 
 
Serum SGPT (U/L) 
 
 
 
Serum cholesterol (mg/dl) 
 
 
 
Serum TG (mg/dl) 
 
 
 
Serum HDL (mg/dl) 
 
 
 
Serum LDL(mg/dl) 
 
 
 
Serum FBS (mmol/L) 
 
 
 
Serum HbA1c (%) 
 
 
 

 
 
39 
 
Modified borg scale: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SCALE SEVERITY 
0 
No Breathlessness At All 
0.5 
Very Very Slight (Just Noticeable) 
1 
Very Slight 
2 
Slight Breathlessness 
3 
Moderate 
4 
Somewhat severe 
5 
Severe Breathlessness 
6 
 
7 
Very Severe Breathlessness 
8 
 
9 
Very Very Severe (Almost Maximum) 
10 
Maximum 

 
 
40 
 
 
AppendixIII 
 
Standard pharmacotherapy for stable patient of ACO: 
                                                                              (GOLD 2015) 
 Inhaled corticosteroid (in low or moderate dose, depending on level of  
symptoms)                                              
 Inhaled long acting bronchial agonist (LABA), and /or  
 long acting mascarinic agonist (LAMA)  
 Advice- 
        - on smoking cessation. 
        - Appropriate self management strategies. 
        - Regular follow up.   
                
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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AppendixIV 
Cholecalciferol supplementation schedule for D3 deficient [serum 25(OH)D<30 
ng/ml] ACO pts 
Our aim is  
 to avail (within 6 months) and maintain serum 25(OH)D @ 60-80 ng/ml + 
serum Ca2+ @ 8.5-10.5 mg/ml,  
 for having extra-calcaemic effect of vitamin D3,  
 on lungs and other tissues 
At 1st visit / at day 0: 
Cholecalciferol 80,000 IU (2 capsules of 40,000 IU) / wk, for 12 weeks (3 months)  
At 2nd visit / at day 90 / after 3 months: 
If,  
serum 25 (OH)D   
+  
serum Ca2+ = 
Then,  
for next 3 months,   
prescribed dose will be - 
So,  
for next 3 months,   
dose schedule will be - 
30-40 ng/ml  
+ 
8.5-10.5 mg/ml 
4600 IU/day 
   4600 IU X 7  
= 32,200 IU / 7 days   
= 1 cap (40,000 IU) / wk 
40-50 ng/ml 
+ 
8.5-10.5 mg/ml 
3000 IU/day 
   3000 X 15 
= 45,000 IU / 15 days 
= 1 cap (40,000 IU) / 2 wks 
60-80 ng/ml 
+ 
8.5-10.5 mg/ml 
not suggested in guideline, but, to 
get extra-calcaemic effect of D3 
   1 cap (40,000 IU) / 6 wks 
 
> 80 ng/ml   
+ 
8.5-10.5 mg/ml 
stop taking drug & symptom 
analysis 
   no dose for 3 months 
 
>150 ng/ml  
+  
>10.5 mg/ml 
 
close monitoring and ask for –  
 feeling sick or being sick 
 poor appetite or loss of 
appetite 
 feeling very thirsty  
 passing urine often 
   no dose for 3 months 
 

 
 
42 
 
 constipation or diarrhea  
 abdominal pain 
 muscle weakness  
 pain 
 bone pain 
 feeling confused 
 feeling tired 
At 3rd visit / at day 180 / after 6 months: 
Pt will be referred to physician and will be suggested to follow the above-mentioned 
schedule 
Ref:  Vitamin D Council. I tested my vitamin D level. What do my results mean? 
2017 
[Internet]. 
[Cited 
2018 
March 
25]. 
Available 
from: 
https://www.vitamindcouncil.org/i-tested-my-vitamin-d-level-what-do-my-results-
mean/  
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
43 
 
Appendix-V 
Principles of Bangladesh Good Clinical Practice  
                                 (Directorate general of drug administration 2011)                
Before conducting the study the researcher will follow all the principals of 
Bangladesh GCP, written below: 
1. Clinical trials should be conducted in accordance with the ethical principles that 
have their origin in the Declaration of Helsinki, and that are consistent with GCP and 
the applicable regulatory requirement(s).   
 2. Before a trial is initiated, foreseeable risks and inconveniences should be weighed 
against the anticipated benefit for the individual trial participant and society. A trial 
should be initiated and continued only if the anticipated benefits justify the risks.   
 3. The rights, safety, and well-being of the trial participants are the most important 
considerations and should prevail over interests of science and society.   
 4.The available nonclinical and clinical information on an investigational product 
should be adequate to support the proposed clinical trial.   
 5. Clinical trials should be scientifically sound, and described in a clear, detailed 
protocol.   
 6. A trial should be conducted in compliance with the protocol that has received 
prior institutional review board (IRB)/ independent ethics committee (IEC) approval/ 
favorable opinion.  
7. The medical care given to, and medical decisions made on behalf of, participants 
should always be the responsibility of a qualified physician or, when appropriate, of 
a qualified dentist.  
 8. Each individual involved in conducting a trial should be qualified by education, 
training, and experience to perform his or her respective task(s).   
 9. Freely given informed consent should be obtained from every participant prior to 
clinical trial participation.   
 10. All clinical trial information should be recorded, handled, and stored in a way 
that allows its accurate reporting, interpretation and verification.   

 
 
44 
 
 11. The confidentiality of records that could identify participants should be 
protected, respecting    the    privacy and confidentiality rules in accordance with the 
applicable regulatory requirement(s).   
 12. Investigational products should be manufactured, handled, and stored in 
accordance with applicable good manufacturing practice (GMP). They should be 
used in accordance with the approved protocol.   
 13. Systems with procedures that assure the quality of every aspect of the trial 
should be implemented 
. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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