a School of Dietetics and Human Nutrition, McGill University, 21, 111 Lakeshore Road, Ste Anne de Bellevue, Quebec H9X 3V9, Canada. E-mail: tkassa{at}po-box.mcgill.ca
b Department of Epidemiology and Biostatistics, 1110 Pine Avenue West, McGill University, Montreal, Quebec H3A 1AB, Canada.
c Department of Nutrition, University of Montreal, C.P. Succursale centre ville, Montreal, Quebec H3C 3J7, Canada.
Abstract
Background In developing countries, studies using morbidity recalls to evaluate the benefits of vitamin A on respiratory health in children under 6 years of age have been inconclusive. This relationship has not been examined in older children. Spirometric measurements, an objective means of assessing respiratory health, require the subject's collaboration and have been successfully used in children over 6 years of age. This report describes a cross-sectional analysis of the relationship between lung function and vitamin A status in an area endemic to vitamin A deficiency.
Methods The data on which this report is based were gathered prior to the implementation of a prospective trial of the effect of vitamin A supplementation on lung function level in Northern Ethiopia. Vitamin A status was assessed by the Modified Relative Dose Response (MRDR) method and lung function assessed by spirometry in 702 rural children aged 69 years. Demographic, personal health, household, environmental and socioeconomic data were gathered by questionnaire.
Results In children with low vitamin A reserve, the unadjusted forced expiratory volume in one second (FEV1) was 48.8 ml (P = 0.006) lower than in those with adequate reserve. This difference was 23.1 ml (P = 0.04) when adjusted for age, gender and height and 14.1 ml (P = 0.20) when adjusted for children's demographic, general health, lung function and household-related characteristics.
Conclusion Although these findings suggest that vitamin A plays a relatively minor role in determining FEV1 level, interpretation is limited by the cross-sectional design. Further clarification of its role requires a trial of vitamin A supplementation.
Keywords Vitamin A deficiency, vitamin A reserve, modified relative dose response, MRDR, lung function, FEV1, Ethiopia
Accepted 30 May 2000
As early as 1925, experimental studies in animals had convincingly shown that the respiratory system is a target of vitamin A action.1 After the onset of vitamin A deficiency, the mucus-secreting epithelium was replaced by stratified squamous keratinizing epithelium in the trachea and bronchi of vitamin A deficient rats. Autopsy studies in vitamin A deficient children have also demonstrated keratinizing metaplasia of the respiratory tract.2 Animal studies also suggest that the morphological changes observed in vitamin A deficiency predispose the development of infection3 and the changes observed during vitamin A deficiency have been shown to disappear in a time-dependent fashion when dietary retinol was restored.4 Similar results have been reported in more recent and detailed studies.57 In vivo hamster trachea studies confirmed that replacement of the normal mucus by squamoid cells could result in the near occlusion of the tracheal lumen.5 Tracheal ring size of vitamin A deprived hamsters was also found to be significantly reduced compared to that of the controls.6
In developing countries, observational studies in children have shown an increased risk of acute respiratory infections associated with vitamin A deficiency.810 However, in a meta-analysis assessing the effectiveness of vitamin A intervention in children 6 months to 6 years of age, there was non-conclusive evidence on morbidity, especially from respiratory infections.11 Some studies (using morbidity recalls) have even suggested that vitamin A intervention with large doses of vitamin A may increase the number of respiratory infections.12,13 This was attributed to the stimulating effect of vitamin A on a healthy respiratory epithelium.14
A Medline search for the period January 1966 to December 1999 did not identify any reported study addressing the association of vitamin A status and lung function level in children. However, five studies (two cross-sectional and three prospective) were identified in adults. The results from the two cross-sectional studies were conflicting. In the First National Health and Nutrition Examination (NHANES I), in a subsample of 1510 subjects aged 2574 years, a significant inverse relationship between dietary vitamin A intake (24-hour recall) and airway obstruction (defined by FEV1/FVC 65%) was found after adjusting for age, sex, body mass index (BMI), caloric intake and cigarette smoking.15 However, in the Atherosclerosis Risk in Communities (ARIC) study in 15 743 subjects aged 4564 years, vitamin A intake (assessed by food frequency questionnaire) was not related to airway obstruction in any smoking categories using either categorical or continuous lung function measurements (FEV1 [forced expiratory volume in one second], FVC [forced vital capacity], FEV1/FVC) with the exception of current smokers in the upper quartile group.16 By contrast, all the three short-term intervention studies1719 found significant improvement in lung function levels. In the first study, a randomized controlled trial (RCT) of 30 human volunteers with adequate vitamin A status and long history of smoking were given 25 000 IU vitamin A or placebo daily over a 30-day period and a 15.1% increase in FEV1/FVC was recorded in the supplemented group.17 In the second RCT study, 12 male patients with mild chronic obstructive pulmonary disease (COPD) were treated with vitamin A (1000 RE) or placebo over the same period and a 22.9% increase in FEV1 was obtained in the vitamin A treated group.18 In the last study, 816 asbestos-exposed subjects with high rate of smoking were entered (in the pilot Carotene and Retinol Efficacy Trial [CARET] and they exhibited a 70 ml increase in FVC (P < 0.05) over the predicted value when serum retinol concentration increased from the 25th to the 75th percentile.19
The objective of the present study was to describe the association between lung function and vitamin A status (biochemical assessment) in children 69 years of age in an area endemic to vitamin A deficiency. The data were gathered to provide baseline information prior to the implementation of a RCT to evaluate the effect of vitamin A supplementation on their lung function status.
Methods
Details on site and population selection are given in our previous report20 which describes the prevalence of clinical and subclinical vitamin A deficiency. Briefly, the study was carried out in five study sites (Mesanu, Genfel, Abreha-Atsbeha, Negash and Gemad) in Wukro wereda, Tigray administrative region, Northern Ethiopia from April to July 1997. The altitude of these sites ranged from 1900 m to 2330 m above sea level. The study population comprised 1339 children, 69 years of age, randomly selected from the five study sites with proportional probabilities to their respective population sizes. Oral informed consent was obtained from parents. Ethical approval was obtained from the Ethiopian Science and Technology Commission, the Ethics Committee of the Department of Pediatrics and Child Health, Addis Ababa University, Ethiopia and the Ethical Review Committee of Macdonald Campus of McGill University, Canada. Methods of measurement described below included a questionnaire (answered by the mother or female caregiver), anthropometry and assessment of vitamin A status based on ophthalmological examination, dietary vitamin A intake, biochemical assessmentmodified relative dose response (MRDR)21,22 and serum retinol as part of the MRDR assay.
Questionnaire
The study questionnaire was developed by combining questions from the American Thoracic Society (ATS) questionnaire on respiratory health status, a food security study questionnaire previously used in the area, and questions specifically related to this study. It included questions related to the child's health and to the household and to the environmental and socioeconomic characteristics of the home. It was translated to the local language, Tigrigna, and pretested before use.
Anthropometry, vitamin A assessments and lung function tests were performed starting within 2 weeks of the completion of the questionnaire in four health facilities (Agulae Health Station, Wukro Junior Hospital, Abreha-Atsbeha Health Station and Negash Clinic) serving the five study sites.
Vitamin A assessment
The Modified Relative Dose Response (MRDR) developed by the Vitamin A Research Group at Iowa University was used to assess the liver vitamin A reserve. Serum retinol was determined as part of the MRDR assay. A cutoff of MRDR ratio of 0.06 identified children with low vitamin A reserve (subclinical vitamin A deficiency) and serum retinol levels of
0.35 µmol/L and between 0.36 µmol/L to 0.70 µmol/L (inclusive) defined clinical and subclinical vitamin A deficiency, respectively.
Ophthalmolgical examination for xerophthalmia was conducted without differentiation of the various stages of xerophthalmia. Children exhibiting ocular signs were immediately given vitamin A capsules and were therefore not eligible for the intervention study. The data on these children were used as reference values for vitamin A deficiency in the present study.
Lung function tests
At the health facility, lung function tests were performed using two portable Microlab 3300 spirometers (Micro Medical Ltd, Chatham, Kent, ME4 4TQ). Children were tested in groups of five. The technician explained and demonstrated lung function measurement. Each child then performed the spirometric measurements in the standing position with the use of a noseclip, according to the guidelines of the American Thoracic Society.23 The values were corrected to BTPS (body temperature, barometric pressure and fully saturated). A minimum of three reproducible results were required for each child and up to eight attempts were permitted. Values for forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were required to be reproducible within 5% for at least two of the three acceptable spirograms. Reproducibility and acceptability were confirmed by the technician who scrutinized the volume-time spirograms. The best FEV1 and FVC from any acceptable flow-volume curve were used for analysis24 and from these values the ratio FEV1/FVC expressed as a percentage was calculated. All spirograms and demographic data were finally rechecked on site and children who were unable to provide an acceptable spirogram on the first trial repeated lung function tests after at least one hour. The study children stayed within the health facility 46 hour to complete all other assessments and therefore were available for retesting. For consistency, in children who performed two spirogram trials, only the first was used in the analysis.
The child's age was derived from an events calendar. Birth weight data were not available. Height and weight were measured prior to spirometry and gender recorded. In order to ascertain the negative effects of recent respiratory illness on lung function, parents were asked whether their children experienced cough during the past month, the past 2 weeks or the past week. The technician performing lung function tests also checked for cough and reported cough at time of spirometry. Spirometry was conducted even if a child had a cough, as rescheduling of children was not possible for logistic reasons.
Data analysis
Data were entered using Epi-Info program and analysed using the SAS system for Windows (version 6.11; SAS Institute, Cary, NC). Statistical significance was set at P < 0.05. Differences between study groups were assessed by Student's t-test and 2 tests for continuous and categorical variables, respectively. Standardized nutritional indices were calculated with height, weight and gender data using Epi-Info program (version 6.02, CDC, USA and World Health Organization [WHO], Geneva, Switzerland). Children were classified as underweight, stunted and wasted when the weight-for-age, height-for-age and weight-for-height Z-scores were below 2.0 standard deviations (SD), respectively, using the United States National Center for Health Statistics (NCHS) as the reference population.
As a first step in the analysis, the crude association between FEV1 (ml), the primary outcome variable, and all potential explanatory variables were evaluated. The analysis was also repeated with FVC and FEV1/FVC as outcome variables. The main explanatory variable was vitamin A status determined by the MRDR method because it was a specific and reliable method that assessed liver vitamin A reserve. Partial (age-gender and height) and full adjustments for possible confounders were done using multiple linear regression. Explanatory variables were included in the multiple regression model based on statistical significance (P 0.2) or substantive knowledge. Exceptions to this were symptoms such as breathlessness when playing in comparison to other children of the same age (P = 0.01); usual cough during the past year (P = 0.0001); chest sounding wheezy (P = 0.13). Although these variables were eligible for inclusion based on statistical significance, they were not included in multiple linear regression as they were considered possible outcomes of a reduced vitamin A reserve.
Results
Characteristics of study population
Of the 792 eligible children, 702 (88.6%) had complete data for vitamin A status, anthropometry, lung function and its associated risk factors, and constitute the population for the present study. Table 1 compares the personal and household characteristics of the children with complete (n = 702) and incomplete (n = 90) data. Those with incomplete records have statistically significantly lower indices of vitamin A status than those with complete data. They were also likely to come from female headed households and there were between-site differences. However, the lung function measurements were not different between the two groups.
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Personal characteristics and anthropometry
Of note were the gender differences, with FEV1 in girls being 49.9 ml lower than in boys (P = 0.004). Also for each increase in age, FEV1 increased by 123.2 ml (P = 0.0001) while for each increase in birth order FEV1 was 8.1 ml lower (P = 0.22). FEV1 in children who attended school was 218.1 ml greater than FEV1 in those who did not (P = 0.0001). By contrast, the difference between children who were breastfed for more than 2 years was modest (26.8 ml) and the strength of association was less (P = 0.13). Age, height, weight and BMI were also strongly positively associated with FEV1. In children who were underweight or stunted, FEV1 was 202.7 ml and 213.8 ml lower, respectively, than their referents (P = 0.0001).
Health-related variables
Among the health-related variables associated with FEV1, children who had had measles had FEV1 levels 28.9 ml higher than their referents (P = 0.10). Conversely, children who were vaccinated had FEV1 values 88.7 ml lower than those non-vaccinated (P = 0.0001). Children who resided in areas where the distance to the nearest health facility was more than one hour had FEV1 29.5 ml lower compared with their referents (P = 0.10).
Several respiratory related variables not shown here were also associated with lower lung function level. These variables were: (i) breathless when playing in comparison to other children of the same age (64.4 ml lower FEV1; P = 0.01) and (ii) usual cough during the past year (135.9 ml lower FEV1; P = 0.0001). Children for whom cough was reported during the month prior to lung function measurement or whose chest sounded wheezy had FEV1 45.7 ml and 37.0 ml lower, respectively, than their referents (P = 0.14 and P = 0.13, respectively).
Among the technical factors which may contribute to between-individual variation in spirometry, only technician difference was found to be significantly associated with lung function level (data not shown).
Household-related factors (data not shown)
Children from households with fewer than six members had FEV1 27.8 ml lower than their referents (P = 0.11) but none of the other household factors measured were eligible for inclusion in the analysis based on P 0.2. Thus no relationship was found between the lung function level of the child and literacy status of the mother or living in houses with one versus more than one room. Children who (as infants) were carried on the back by mothers while cooking had values 26.9 ml lower than their referents (P = 0.40) and there was no association between FEV1 and living in a house that had a kitchen attached to the main house versus not attached.
Partially and fully adjusted results
Variables which showed significant (P < 0.05) or marginal crude associations (P 0.2) with FEV1 or were considered to be important on a priori grounds were included in a multiple linear regression model to assess the independent effect of vitamin A status on lung function level (Table 2
). Those included on a priori basis were vitamin A status, gender, height, BMI, age, birth order, severe chest illness before 2 years of age, cough during the month prior to the lung function tests, altitude, technician difference and spirometer difference. The determinants were also partially adjusted for age, gender and height, prior to full adjustment.
As expected, results of the partially (age-gender-height) adjusted values were intermediate between the crude and fully adjusted values. The potential effect of vitamin A status on FEV1 diminished from 48.8 ml to 23.1 ml (95% CI : 45.2, 0.9) when partially adjusted for age, gender and height and to 14.1 ml (95% CI : 35.7, 7.5) after fully adjusting for the independent variables listed in the footnote to Table 2. After adjustment personal factors, gender, height and BMI still showed strong significant association with level of FEV1. For each increase in birth order, FEV1 was 12.1 ml (95% CI : 21.9, 2.3) lower while those children who attended school had a mean FEV1 26.0 ml (95% CI : 18.8, 70.6) higher than their respective referents. Two variables, underweight and stunting which showed significant association in the bivariate analysis, were not included in multiple linear regression analysis because both were strongly correlated with each other and with height. Similarly, weight was not included in the model because it was strongly correlated with height (BMI was included instead).
There was no relationship between FEV1 and the health-related variables nor with symptoms of respiratory illness (Table 2). Among the lung function related variables, technician difference was strongly associated with FEV1 and spirometer only marginally associated with lung function level.
Table 3 shows the relative contribution of each determinant to the adjusted association between vitamin A status and lung function level. Columns (1) and (2) show the distribution of the various FEV1 determinants in children with adequate and low vitamin A reserve, respectively, and column (3) the difference between them. For example, while the adequate and low vitamin A reserve groups are similar with respect to age, there were substantial differences with respect to other determinants, e.g. height, percentage breastfed for more than 2 years, percentage with severe chest illness before 2 years of age. Column 4 shows how influential each of the determinants is on FEV1. The last column which is the adjustment in FEV1 required for each determinant is calculated from the product of columns 3 and 4. The total adjustment, 34.7 ml, was obtained by summing the values in this last column. This total adjustment was then deducted from the crude effect of vitamin A on FEV1 (48.8 ml) to get the net effect (adjusted difference) of vitamin A on FEV1 (14.1 ml), an estimate which corresponds to the value of 14.1 ml obtained by multiple linear regression (Table 2
). Note that largest adjustment of 21.9 ml FEV1 (63.1% of the total adjustment) was necessitated by the 0.97 cm height deficit in the low vitamin A reserve group compared to the vitamin A adequate group.
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Discussion
We initially found a strong, crude association between lung function and vitamin A status with FEV1 being 48.8 ml lower in children with low vitamin A reserve. However, the age-gender-height adjusted effect was reduced to 23.1 ml (95% CI : 45.2, 0.9) and when fully adjusted for determinants including personal, health-related factors, respiratory history, household factors and technical factors related to lung function measurement, the strength of association was further reduced (adjusted effect = 14.1 ml (95% CI : 35.7, 7.5). Girls, as expected, had FEV1 values 34.8 ml lower than boys25,26 (95% CI : 56.2, 13.4). Also, as expected, height and BMI were strongly and positively associated with FEV1 (P < 0.05) but age was not (P = 0.21). Birth order (treated as a continuous variable) was strongly, negatively associated with lung function level, with FEV1 lower by 12.1 ml (95% CI : 21.9, 2.3) for every older sibling the child had. Differences between technicians in carrying out tests are a well-recognized source of between- and within-individual variation in lung function testing27 and the size of effect shown in our study (38.6 ml, 95% CI : 62.7, 14.5) indicates the importance of taking this into account in any study using more than one technician.
In our study, spirometry was conducted even if a child had recent respiratory symptoms as rescheduling was not possible. Although the proportion of children who had recent cough was greater in the low vitamin A status group (0.55) compared to those with adequate vitamin A reserve (0.45), adjustment for this variable resulted in minimal change (Table 3).
The direction of some of the associations between health-related variables (vaccination and measles status) and FEV1 may seem surprising. For instance, having had measles versus not was associated with higher FEV1 level (unadjusted 28.9 ml). This is in keeping with the evidence that measles infection (but not measles inoculation) may protect against asthma,28 a condition characterized by airflow limitation and reflected in lower lung function levels (FEV1, FEV1/FVC). Likewise, having had no vaccination (versus any) was associated with a higher FEV1 level (unadjusted 88.7 ml). With adjustment, neither of these remained significant.
In our study the factors which may have diminished the ability to detect an association between vitamin A and lung function need to be considered. For instance 9.4% of the children with low vitamin A reserve were not included on account of incomplete records (Table 1). Though this may have weakened the relationship between vitamin A status and lung function, the mean FEV1 in the two groups was similar. Our a priori expectation was that vitamin A affects lung function level through a direct effect on the lungs. It is, however, possible that the vitamin A effect could be mediated by its effect on linear growth which is a known determinant of lung size and lung function and accounted for 63.1% of the total adjustment (Table 3
). Of interest is a recent report of a RCT conducted in 1405 Indonesian children aged 647 months in which vitamin A supplementation was shown to improve their linear growth but the impact was muted with increasing levels of respiratory infection.29 Thus in our study, there was a difference of 0.97 cm in height between the children with adequate and low vitamin A reserve groups and if this was as a result of vitamin A deficiency, adjustment for height would not be appropriate. However, given the cross-sectional design of this study and the absence of information on caloric intake, the height difference may also be due to other nutritional inadequacies associated with low caloric intake (protein, fat, zinc, etc.). To adjust for some of the nutritional status difference, height was replaced with stunting in the model previously used since stunting was not correlated with age and BMI. In this analysis, children with low vitamin A reserve had FEV1 20.7 ml lower than those with adequate vitamin A reserve (P = 0.11). Although statistical significance was not achieved, the estimated effect of vitamin A on FEV1 improved from 14.1 ml to 20.7 ml. However, the model containing stunting explained only 48% of the total variation, whereas the model containing height explained 63%.
The use of an events calendar may have produced imprecision in the measurement of age and therefore diminished the ability to detect an age effect.
In conclusion, the findings of this study suggest that even in an area endemic to vitamin A deficiency, vitamin A status plays a relatively minor role in determining FEV1 level. However, interpretation should be guarded given the cross-sectional design of our study and a more definitive answer may be provided by an intervention of vitamin A supplementation.
Acknowledgments
This study was carried out with the aid of a core grant from the International Development Research Centre (IDRC), Ottawa, CanadaGrant 951052/02746 and partial funding from the Rockefeller Foundation (African Dissertation Internship Award to TK). Dr Mesfin Minas, Head, Bureau of Health, Mekelle, Tigray is highly appreciated for the logistics and moral support he gave during the data collection phase. The Relief Society of Tigray (REST) is acknowledged for its support of the project. Dr Hagos Beyene, former head of Department of Pediatrics and Child Health, Addis Ababa University provided valuable advice during the data collection period. Ms Grace Gerardi and Ms Sheila Saralegui, former staff of Respiratory Epidemiology Unit, Department of Epidemiology and Biostatistics, McGill University are gratefully acknowledged for the intensive lung function measurement training and advice they provided to TK.
Notes
NB. Reprints will not be available from the authors.
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