Vitamin D deficiency during chronic obstructive pulmonary disease exacerbations

Background Vitamin D deficiency is widespread and associated with increased risk of chronic diseases. The relation between chronic obstructive pulmonary disease (COPD) and vitamin D is complex owing to comorbidities, which are affected by vitamin D. Patients with low vitamin D showed higher risk for exacerbations. Aim The aim was to study the status of vitamin D in patients during exacerbation of chronic obstructive pulmonary disease (AECOPD). Patients and methods The study included 205 patients presented with AECOPD and 150 controls. Patients and controls were subjected to full clinical history and examination, pulmonary function testing, and vitamin D [hydroxyvitamin D, 25(OH)D] examination by Liaison 25 OH Vitamin D assay (direct competitive chemiluminescence immunoassay, DiaSorin Inc, Stillwater, Minnesota, USA) in serum. Symptom scoring was done using modified Medical Research Council (mMRC) and combined assessment of Global Initiative for Chronic Obstructive Lung Disease COPD classification, with division into groups A, B, C, and D. Data related to severity of exacerbation, site of care, and hospital days were gathered. Statistics Pearson’s χ2-test was used to compare the prevalence of categorical variables between patients with COPD and control groups. t-Test was used to compare differences in the levels of continuous variables between the two groups. R2-test was used to measure how close the data are to be fitted in the regression line. Results 25(OH)D was significantly lower in patients with AECOPD than control group (mean: 39.5±32.5 vs. 56.3±43.7 nmol/l, P˂0.05). Vitamin D insufficiency (25–75 nmol/l) was significantly higher in patients than controls [115 (56.09%) patients vs. 51 (34%) controls, P<0.05]. Dyspnea score (mMRC) was higher in deficiency group (70.1% having two or more mMRC score) compared in insufficiency and sufficiency groups (51.3 and 51.5%, respectively). Patients with mild and moderate COPD (forced expiratory volume in first second >50%) showed higher 25(OH)D (69.4±23.1) than patients with severe and very severe COPD (forced expiratory volume in first second <50%) (47.4±28.3), with P value less than 0.05. Patients required hospitalization showed lower levels of 25(OH)D compared with patients treated at home (23±14.9 and 52±22.1, respectively), with P value less than 0.05. Hospital days were higher in deficiency group (3.78±3.51 days) compared with insufficiency group (1.68±2.33 days) or sufficiency group (1.3±1.7 days), with P value less than 0.05. Conclusion Vitamin D is low in patients with COPD during AECOPD. The relationship is linear with lung function, disease severity groups and with previous exacerbation rate. Severe exacerbations requiring hospital admission and lengthy hospital stay were demonstrated in patients with low vitamin D.


Introduction
Vitamin D sources originate from skin after exposure to ultraviolet rays, dietary resources, and supplementation [1]. Vitamin D plays a role in bone maintenance and calcium and phosphorous homeostasis, strengthens the innate immune responses upon infection, and regulates the adaptive immune responses [2]. Vitamin D deficiency is widespread worldwide and is associated with increased risk of cancer, autoimmune diseases, infectious diseases, and cardiovascular diseases [3]. Factors affecting vitamin D level include old age, female sex, habits of exposure to sun, dietary nature, and vitamin D supplementations [4]. Vitamin D deficiency role in chronic obstructive pulmonary disease (COPD) is not clear. Some studies linked vitamin D deficiency and COPD [5,6], without definite conclusion. COPD exacerbations affect the disease by symptom worsening and functional and clinical deterioration [7]. Vitamin D deficiency studies showed effect on asthma [8,9], COPD [10], and recurrent respiratory tract infections [11,12]. These studies highlighted the association between vitamin D deficiency and liability to infection, through influence on airway colonization [13] or alteration of airway remodeling [14]. These studies lack prospective clinical trials that study vitamin D during COPD exacerbations.

Aim
The aim was to explore the status of vitamin D in patients with COPD during exacerbation of chronic obstructive pulmonary disease (AECOPD).

Ethics
Written consent was obtained from all patients and controls before inclusion in the study. The study design was approved by the institutional review board.

Participants
Patients with COPD exacerbation who were managed in either pulmonology clinic, Emergency Department, or Internal Medicine Clinic in four referral medical centers were selected for the study prospectively from January 2014 to June 2016. Overall, 205 patients with COPD with AECOPD and 150 controls attending the medicine clinic were selected for the study. All participants were studied for serum 25 hydroxyvitamin D [25(OH)D]. All of them were assessed for demographic data, smoking history, years since diagnosis of COPD, and medications used. All patients and controls were screened for comorbidities. Symptom scoring by modified Medical Research Council Dyspnea Scoring (mMRC) was done. Clinical examination included vital signs and BMI. Patients were further classified per WHO criteria into obese (30 kg/m 2 or more), overweight (25-29.9 kg/m 2 ), normal (18.5-24.9 kg/m 2 ), or underweight (<18.5 kg/m 2 ). Clinical assessment of severity of disease, years since diagnosis of COPD, previous exacerbations, and if there was previous admission owing to COPD exacerbation were recorded. Exacerbation history was defined as having respiratory symptoms treated with antibiotics and/or oral steroids and/or hospitalization in the last 12 months. Pulmonary function tests were retrieved from previous examination, and the ratio of forced expiratory volume in first second (FEV1)/forced vital capacity less than 70% was essential to diagnosis. FEV1% of predicted was recorded, and spirometry was repeated after recovery from exacerbation. Classification of COPD severity by FEV1% from predicted, symptom scoring by mMRC, and risk of exacerbation into group A, B, C and D. Division of patients with COPD into subgroups was done according to the current global initiative for obstructive lung disease (GOLD) classification 2015 [15]. Chest radiography and routine laboratory examinations were done to detect comorbid conditions including diabetes, hypertension, renal failure, and congestive heart failure. Site of care (home treatment, hospitalization, or intensive care) was determined per severity and recorded for each patient. Duration of hospitalization stay was recorded if any for patients who were hospitalized. Arterial blood gas analysis and categorization was recorded as normal blood gases, type I or type II respiratory failure.
The medical records of the patients were collected for retrieving the previous history including pulmonary function tests, previous exacerbations, and previous hospital admissions, and chronic medications used.
Inclusion criteria included all patients diagnosed with AECOPD with previous spirometry showing ratio of FEV1/forced vital capacity less than 70%, and smoking history of at least 10 pack-years. Exclusion criteria included enrollment in other studies, refusal to participate in the study, inability to undergo spirometry, presence of other pulmonary disorders including asthma bronchiectasis or lung fibrosis, renal impairment or failure, presence of osteoporosis, or treatment with vitamin D supplements.

Laboratory analysis
Vitamin D [25(OH)D] in serum was analyzed using the Liaison 25(OH) Vitamin D assay (direct competitive chemiluminescence immunoassay). Samples were stored at 2-8°C for less than 24 h if not tested immediately. Categorization of results per levels supplied by laboratory cutoff values was done into sufficient (<75 nmol/l), insufficient (25-75 nmol/l), and deficient (>25 nmol/l).

Statistical analysis
Pearson χ 2 -test was used to compare the prevalence of categorical variables between patients with COPD and control groups (Table 1). t-Test was used to compare the differences in the levels of continuous variables between TWO groups (e.g. participants with vitamin D sufficiency and insufficiency and insufficiency and vitamin D deficiency based on laboratory cutoff values) in Table 2. R 2 was used to measure how close the data are to be fitted on the regression line. The study also measured the coefficient of determination or the coefficient of multiple determination for multiple regression. It was performed to analyze the relation between vitamin D status and demographics (age, BMI, mMRC, and smoking index). It was also performed to study the relation between vitamin D and severity of COPD disease as determined by COPD GOLD classification, postbronchodilator FEV1, previous exacerbations, and hospital stay days. P value less than 0.05 was assumed to indicate statistical significance of observed associations.

Patient characteristics
Among 376 patients selected for the study, 171 patients were excluded owing to lack of follow-up or inability to perform spirometry after recovery from AECOPD, presence of other pulmonary disorders upon further radiological studies, renal impairment, or the current use of vitamin D supplements. The characteristics of the study population (205 patients and 150 controls) are shown in Table 1. The mean age was higher in the COPD group by 4.5 years but was statistically insignificant. Men were more than women in both COPD and control groups (84.8 and 80.6%, respectively). There was no significant difference in smoking index between current smokers and exsmokers in both patient and control groups, but there were no never smokers in the patient group compared with the control group ( Table 2. Figure 1 showed a linear relationship between vitamin D and age (Fig. 1a) and smoking index ( Fig. 1d) with P value less than 0.05 but there was no relation between vitamin D and BMI (Fig. 1b) and dyspnea severity (Fig. 1c).
There were statistically lower levels of 25(OH)D (62.3 ±20.4) in GOLD group B compared with group A vs. 32.5±18.2, respectively), with P value less than 0.05 as shown in Table 3. Figure 2 showed a linear relationship between GOLD COPD combined assessment classification and vitamin D levels (2A) and FEV1 (Fig. 2b), previous number of exacerbations (Fig. 2c), and total hospitalization days (Fig. 2d). Table 4 shows the subdivision of patients with COPD by vitamin D status into sufficient (>75 nmol/l, n=33), insufficient (25-75 nmol/l, n=115), or deficient (<25 nmol/l, n=57). Patients with vitamin deficiency were

Discussion
The main finding in this study is the lower levels of vitamin D seen during acute exacerbation of COPD compared with controls. AECOPD was defined as worsening or new symptoms from stable daily state that requires additional treatment [16]. Many published studies [17,18] have looked for a relationship between vitamin D level and the risk of high exacerbations rate based on number and severity of previous exacerbations. This study, to our knowledge, is the first prospective study that provides information about the actual status of vitamin D during the different severities of exacerbation of COPD.
Vitamin D levels in patients with COPD were studied before, and many studies showed deficiency of vitamin D [10,19,20] among different groups of COPD, and many other studies indicated higher risk of COPD in vitamin Ddeficient patients [21,22,23,24], indicating a causal and conditional relationship. Heulens et al. [25] in their experimental study on mice found that vitamin D deficiency resulted in increased pulmonary inflammatory mediators and acceleration and aggravation of development of COPD features.
Vitamin D has antimicrobial antibacterial and antiviral effects through different mechanisms, one of them is the control of activity of cathelicidin which is an antimicrobial polypeptide. Cathelicidin was shown to be active against mycobacteria and against other organisms causing COPD exacerbations including antibiotic resistant strains such as Pseudomonas aeruginosa, Staphylococcus aureus, chlamydia, and other groups of viruses [26,27,28]. Such mechanisms could be an explanation for the increased exacerbations and severity of exacerbation associated with low vitamin D in the groups studied.
In a very recent meta-analysis and systematic review of individual participant data in 2017 done by Martineau et al. [29], a significant protective benefit of vitamin D supplementation was seen in prevention of acute respiratory tract infections especially in patients with vitamin D deficiency. In the first published randomized control study [30] in 2012 on vitamin D supplementation on 182 patients with COPD, although there was improvement of the phagocytic action of monocytes in the vitamin D supplement group, it failed to show a significant reduction in the rate of exacerbation; however, this can be attributed to the small sample size in that RCT and being performed in a single center. Another limitation of this RCT study is being performed on patients with severe COPD already on maximum bronchodilator inhalational therapy, and the added therapeutic effect of vitamin will be difficult to obtain. In that study, although the time to first exacerbation did not differ between treated and control groups, the probability of exacerbation per patient-year was seen to decrease by 43% compared with control group without vitamin D supplementation.
In the primary analysis of our study, only 16% of patients with AECOPD showed sufficient levels of vitamin D compared with 42% in controls, whereas 56.09% of patients with AECOPD showed vitamin D insufficiency compared with 34% in the control group, despite there being no statistical significance in the percentage of deficiency in both groups (27.8% in AECOPD compared with 24% in control group). Zhu et al. [31] in their meta-analysis on 18 studies on vitamin D deficiency in COPD showed that serum levels of vitamin D were lower in patients with COPD, but an earlier meta-analysis done by Zhang et al. [32] collected eight studies on asthma and two studies only on COPD, and it did not show a statically significant difference of vitamin D serum levels in COPD and controls. They mentioned that the limitation to their meta-analysis is the low quality of studies collected and being designed as observational studies. In the analysis of our study, 172 (83.9%) patients during exacerbations showed either insufficient or deficient 25(OH)D levels, whereas deficiency and insufficiency were seen in 58% of the controls. Kunisaki et al. [17] in their original research, which was designed to study the effect of daily azithromycin on exacerbation rate in patients with severe COPD, did a secondary analysis on the baseline vitamin D status from blood samples taken at enrollment and observed their patients for subsequent AECOPD and concluded that in patients with severe COPD, baseline 25(OH)D levels are not predictive of subsequent AECOPD. Although their study included large number of participants (73 patients with COPD), limitations were seen including that the study design was based on baseline 25(OH)D and not on actual 25(OH)D at the time of exacerbation. The second limitation was exacerbation information was collected by phone calls or clinical visits for symptoms with duration of at least 3 days and requiring treatment with an antibiotic or systemic corticosteroid, which may underestimate or overestimate the actual exacerbation rates. Another limitation is that most of their patients had severe COPD and were less ambulant with insufficient exposure to sunlight, hence the expected with lower levels of 25(OH)D. Our study included 205 patients, which demonstrates a cross-section of patients with COPD with different level of severities. Moreover, blood samples were extracted at the same time of patient presentation with AECOPD, which reflects an accurate measurement of vitamin D during the exacerbation.
Low baseline levels of vitamin D levels and association with exacerbations of COPD and mortality were not proved in a study done in Dutch and Swiss primary care settings [33]. In contrast, an Italian retrospective cohort study on previous history of exacerbations and hospitalization and relation with vitamin D deficiency conducted by Malinovschi et al. [34] showed that exacerbation frequency and history of hospitalization was associated with more severe vitamin D deficiency.
A recent Bulgarian study by Mekov et al. [35] on the prevalence vitamin D deficiency in hospitalized patients with COPD exacerbation, showed high prevalence of vitamin D deficiency in admitted patients with AECOPD. Vitamin D deficiency was associated with longer hospital stay a low quality of life, and low lung function (FEV1 <50%), but low levels were not associated with different comorbidities. There are limitations in their study as they included hospitalized patients only, and patients on vitamin D supplements during enrollment were not excluded from the study. In our study, it was noticed that patients with frequent previous exacerbations were associated with lower levels of vitamin D, which reflects basically that higher risk for future exacerbations, or reflect the multifactorial nature of COPD disease, and vitamin D deficiency is only one factor.
A recent meta-analysis and systemic review done by Zhu et al. [18] on 21 previous studies that included including 4818 patients with COPD and 7175 controls showed that lower levels of vitamin were associated with increased COPD risk. They also showed that patients with severe and very severe COPD based on GOLD were associated with lower levels of vitamin D compared with those with moderate COPD, despite the association between low vitamin D levels and risk of exacerbation was not proved by their meta-analysis on three studies meeting the criteria. Our results, which showed that vitamin D levels were directly related to degree of COPD severity and low levels of vitamin D, were associated with the degree of airway obstruction as demonstrated by lower FEV1% in vitamin D deficiency and even more when categorized as COPD groups based on GOLD criteria as A, B, C, and D. Lower vitamin D levels were seen in group B compared with A, and also lower levels in group D compared with group C. Shaheen et al. [36] failed to show a direct relationship between low vitamin D and lung function in adults and in COPD but Monadi et al. [37] showed a direct relationship between lower levels of vitamin D and lower FEV1. Another recent study showed in patients with severe and very severe COPD that vitamin D supplementation was associated with decreased rate of exacerbation and improvement of FEV1 [38]. Moreover, our study showed inverse relation between the number of previous exacerbations and 25(OH)D, and in exacerbations that needed hospitalization, more hospitalization days were inversely related to vitamin D levels; this relationship was also has been demonstrated by other studies [34,35].
In their study on Danish general population including 12 041 individuals, Skaaby et al. [39] found that despite a higher rate of vitamin D deficiency was associated with COPD and mortality owing to COPD, there is no effect on its prevalence, suggesting that vitamin D deficiency is an outcome rather than a cause of COPD. This was also demonstrated in secondary analysis of our study which showed low vitamin D levels were associated with older age and female sex, and this may be related to either disease severity in elderly and in women in our study, or related to lack of exposure to sunlight and activity which is more common in elderly and women in our community. Although smoking pack-years did not show a significant difference between ex-smokers and current smokers in COPD and control, there was a significantly higher mean vitamin D level in current smokers compared which ex-smokers; it could be explained by more severe COPD diseases and older age seen in the ex-smoker group compared with the current smokers in the present study.
Vitamin D was not influenced by weight as measured by BMI in our study, as there was no difference between underweight, normal, overweight, or obese patients. This is in contrast to another study [40] which showed decreased circulatory level of vitamin D in obese people, which reflects the multifactorial effect of COPD disease and its relationship with vitamin D is not simple. Moreover, symptom score in our study measured by mMRC questionnaire did not show a statistical difference between low symptoms (0-1 score) and high symptoms (2 or more) in patients with sufficiency and insufficiency, but 70% of patients with vitamin D deficiency showed high symptoms compared with patients with insufficiency.
In the present study, COPD exacerbations seen in patients with sufficient 25(OH)D were in nature mild and did not require hospitalization, whereas in patients with insufficiency and deficiency, hospitalization and even ICU admissions were seen in most patients; moreover, there was a linear relationship between vitamin D and hospital stay days, as the more hospital days were seen related to low vitamin D. Patients with type I or type II respiratory failure were seen more in patients with deficiency or insufficiency, which in turn suggests that vitamin D is a factor adding to the complexity of disease and aggravating the disease status.
The limitations of our study are that it did not include patients with COPD with no exacerbations to compare their vitamin D levels with patients with AECOPD, and our study did not focus on sunlight exposure. Another limitation is that we did not study the level of vitamin D after recovery from exacerbation to demonstrate any change from the level retrieved at exacerbation.
In conclusion, vitamin D is low in patients with COPD during AECOPD, and the relationship is linear with lung function, disease severity groups, and with previous exacerbation rate. Severe exacerbations requiring hospital admission and lengthy hospital stay were demonstrated in patients with low vitamin D. writing the discussion. Naser K. Abdelnaby was responsible for collection of cases matching with inclusion criteria, collection of results, and analysis of results. Moustafa S. Moustafa was responsible for collecting cases matching with inclusion criteria, collection of results, and analysis of results. Waleed R. Arafat was responsible for collecting cases matching with inclusion criteria, collection of results, and analysis of results.
This work studies the status of vitamin D during COPD exacerbations and whether it is affected by severity of COPD itself or the current severity of COPD exacerbation. The work is conducted in Chest Diseases Department Cairo University.

Financial support and sponsorship
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Conflicts of interest
There are no conflicts of interest.