An Observational and Comparative Study of Diurnal Variation of Spirometry Test Parameters among First and Second Year Normal and Healthy Medical Undergraduate Students

Introduction: This study was designed to assess and compare diurnal variability of FEF25, FEF50, FEF75, FEF25-75, PEF and FEV1 by measuring these parameters during morning and evening hours in normal healthy subjects. Materials and Method: 190 students were enrolled and divided intogroupsof 8-10 students. Each group were directed to appear at different dates in Pulmonary Function Test (PFT) Laboratory at 7:30 AM and again at 5:00 PM for spirometry testing. Spirometry was performed with Spiro Excel 1.1 as per the ATS guidelines and by trained technician. Finally, data from 169 subjects was found to be complete and appropriate and was taken for the analysis.Diurnal variability in FEF25, FEF50, FEF75, FEF25-75, PEF and FEV1 were determined and compared. Results: Allparameters were more in male than female. All the parameters were significantly high in evening tests as compared to morning tests except FVC. Diurnal variability among different spirometry parameters was significantly different (ANOVA, p<0.05) in morning and evening tests. The diurnal variability was highest in large airways as reflected by FEF75 and lowest in smaller airways as reflected by FEF25. The diurnal variability was lowest for FEV1%. It revealed that all parameters exhibit significant diurnal variability. Conclusion: FEV1, FEF and PEF had shown diurnal variability which was directly related to the airway calibre. Greater variability was seen in PEF as compared to FEV1 i.e. proximal airways showed greater diurnal variation than distal airways.


Introduction
Variability in calibre of airways is a normal physiological process in normal personsand this variability may become exaggerated in patients of asthmatic and chronic obstructive pulmonary disease (COPD).Measurement of bronchial hyper-reactivity and airways variability has always posed challenge performing experiments on pulmonary function. Variability in peak expiratory flow (PEF) has been suggested as indicator for bronchial hyper-reactivity. [1][2][3][4][5][6][7][8] The phenomenon of nocturnal asthma has always perplexed clinician's and researcher's mind. Peak expiratory flow rate (PEFR) variability has been suggested as a marker for bronchial hyper-reactivity in asthmatic individuals. [9,10] PEFR variation has been widely advocated and used in clinical practice and asthma research. The National Heart Lung and Blood Institute (NHLBI) and others have recommended, a diurnal variation of 20% or more, as a diagnostic benchmark for asthma. [11,12] Airway function exhibit variability over 24-h periods. This variability has a base that lung function gets worse at night in nocturnal asthma patients and to a lesser extent with COPD. [13][14][15][16] As nocturnal asthma is common and troublesome, [13,14,17] circadian variation in airway function has been of considerable interest in respiratory medicine. It has been recognized that diurnal variation in airway calibre occurs in healthy subjects as well. [18][19][20] It has been suggested that diurnal variation of PEFR in excess of 20% can be used for diagnosis of bronchial asthma in remission where routine spirometry may not show any significant obstructive defect. [19,21] Previous studies mentioned that PEFR shows time to time variation with respect to day and night cycle with specific pattern of lowest at early morning and highest at evening in normal as well as in asthmatics. [6,7,22] PEFR variation has been widely advocated and used in clinical practice and asthma research.
Several evidences suggest that airway variability exhibits a definite circadian pattern in which morning PEF levels are lower than daytime values, with a minimum in early morning and peak in evening. [6,7,22,23] The pattern of variability is exaggerated in smokers and in COPD and in asthmatic patients. [6] The various spirometry indices reflect airflow characteristics of different airways. Forced expiratory flow (FEF), at 25% FVC, i.e. FEF25 reflects small airways, at 75% FVC (FEF75) reflects large airways and at 50% FVC (FEF50) reflects mid/small airways. FEF from 25% to 75% FVC (FEF25-75), reflects mid/ small airways and is also known as mid expiratory flow. Forced expiratory volume in one second (FEV1) reflects the calibre of both large and small airways, whereas PEF is more a reflection of the calibre of large airways. [24,25] In general FEV1 is a more reliable indicator of airflow limitation than PEF. [26] Unfortunately, most studies that describe diurnal variability in airways calibre in asthmatics have used PEF rather than FEV1. Moreover, the diurnal variability of small, mid and large airways has not been studied systematically. This study was designed to assess and compare diurnal variability of FEF25, FEF50, FEF75, FEF25-75, PEF and FEV1 by measuring these parameters during morning and evening hours in normal healthy subjects. Enrolled students were divided into different groups with 8-10 students in a group. Each group were directed to appear at different dates in Pulmonary Function Test Laboratory at 7:30 AM and again at 5:00 PM for spirometry testing. Spirometry was performed with Spiro Excel 1.1 by trained technician between 7:30 to 8.00 AM in morning and 5:00-5:30 PM in evening. PFT was done as per the ATS guidelines [27] The test curve with the highest sum of the FVC and FEV1 were taken for further analysis.

Materials and Method
Recorded data was scrutinized and any incomplete or inadequate test record was rejected. Finally, data from 169 subjects was found to be complete and appropriate and was taken for the analysis.
Statistical Analysis: Paired t-test was used to analyse and compare FEV1, FEF25, FEF50, FEF25-75, FEF75 and FVC values obtained from morning and evening tests of each student. Diurnal variation (dv) i.e. difference between morning and evening values of all parameters for each student were calculated as mean ± SD. The Diurnal variabilities of different parameters were compared using one-way analysis of variance. The statistical analysis was performed by Instat GraphPad Software. A p-value ≤0.05 was considered as significant.

Results
Out of enrolled 190 students,data of 169 students were analysed. Male (n=96) and female (n=73) ratio was 1.32:1. Mean age of all students was 24.48±3.12. Mean height and mean weight of students was 168.22±8.68 and 60.37±10.42 respectively.  All the spirometry parameters were significantly high in evening tests as compared to morning tests except FVC. Diurnal variability among different spirometry parameters was significantly different (ANOVA, p<0.05) in morning and evening tests. The diurnal variability was highest in large airways as reflected by FEF75 and Lowest in smaller airways as reflected by FEF25. The diurnal variability was lowest for FEV1%.

Discussion
Spirometry parameters had shown gender variation. All parameters were more in male than female. Showed a clear evidence that sex is a factor that affects PEF. [28] Spirometry parameters exhibits circadian pattern andthey were less in morning compared to evening time. Diurnal variability may be seen due to variability in airway calibre during morning and evening time. [6,7,22,23] Various studies have shown the diurnal variability of different spirometry parameters like Kondo S, Erban J et al. and Troyanov S et al. had demonstrated that spirometry parameters had significant difference during morning and evening time especially FEV1% and PEF and consistent with the results obtained from present study. [29][30][31] Present study had made an attempt to differentiate the diurnal variability in spirometry test due to change in calibre of proximal and distal airway using PEF and FEV1. Present study results were consistent withthe study done by Hegewald MJ et al., who had exhibited that intrinsic variability in a single session (both morning and evening) spirometry test was higher for PEF than FEV1 also diurnal variability of PEF was higher than FEV1 in healthy subjects. [32] Changes in proximal airway calibre results in changes in PEF while changes in FEV1 is related to calibre of proximal and peripheral airway. [24] Studies have interpreted that the variability in proximal airways is largely due to changes in airway geometry. Fractional reduction in large airway calibre leads to greater decrease in flow compared to smaller airways. And it occurs due to a theory according to that flow rate or resistance is inversely proportional to the fourth power of radius. Also, it is a fact that the proximal and distal airways differ in smooth muscle content and nerve supply. The density of nerve supply and smooth muscle mass decreases as we proceed from proximal to distal airways. [33] This is why the diurnal variability in smaller airways is lower than larger airways.
Correlation between PEF and FEV1 and their diurnal variability was significant. This feature was representation of changes in proximal airways calibre corresponding to changes in distal airways calibre. Morning and evening mean of both PEF and FEV1 were significantly different and showed diurnal variability. Previous study also supported results of this study. [2,6,23] FEV1 was clinically more suitable to know the diurnal variability because total variability was lowest and maximum variability seen was less than 10%. Clinical use of Mid Expiratory Flow was not justified because it showed high variability. FEV1 and PEF showed variability according to the previous study.

Conclusion
FEV1, FEF and PEF has shown diurnal variability which was directly related to the airway calibre. Proximal airways showed greater diurnal variation than distal airways due to in their calibre, reflected by greater variability in PEF as compared to FEV1. In this study only two readings were taken to investigate the diurnal variability. Further study with multiple recordings in 24hour duration should be tried to better characterize the circadian pattern of spirometry parameters and exploring their physiological basis.