To evaluate the effect of antioxidant Vitamins E and C as adjunct therapy of severe acute lower respiratory infection (ALRI) in children.
Randomized double-blind placebo-controlled clinical trial.
A large childrens' hospital serving the urban poor in Kolkata, India.
Children aged 2–35 months admitted with severe ALRI.
In total, 174 children were randomly assigned to receive α-tocopherol 200 mg and ascorbic acid 100 mg twice daily or placebo for 5 days. All children received standard treatment for severe ALRI. Outcome measures were: time taken to recover from a very ill status, fever, tachypnoea, and feeding difficulty; and improvement in oxidative stress and immune response indicated by thiobarbituric acid reacting substances (TBARS) and response to skin antigens, respectively.
Recovery rate ratios (95% CI) using proportional hazards model were 0.89 (0.64–1.25), 1.01 (0.72–1.41), 0.86 (0.57–1.29), and 1.12 (0.77–1.64) for very ill status, feeding difficulty, fever, and tachypnoea, respectively. TBARS values were high and similar in the two groups at admission, discharge, and at 2 weeks follow-up. Serum α-tocopherol significantly increased in treated group at discharge. Immune response to skin antigens were very poor at admission and after 2 weeks, in both groups.
Infants with severe ALRI failed to benefit from two antioxidant nutrients as adjunct therapy. Severe ALRI in infants may cause cell-mediated immune dysfunction. We need a better understanding of oxidative processes in growing infants to help us better design interventions with antioxidant therapy.
Acute respiratory infection (ARI) is the most frequent illness globally and a leading cause of death in the developing world, particularly in infants and young children (Stansfield et al., 1993). Intervention studies in several developing countries with case management algorithm to treat pneumonia in an operational setting found that ARI-specific mortality declined by an average of 42%, whereas overall mortality reduced by an average of 23% (Stansfield et al., 1993). Improvement in the case management of pneumonia may therefore contribute to further reduction in ARI-specific mortality. Two nutrient vitamins α-tocopherol (vitamin E) and ascorbic acid (vitamin C) are well-established dietary antioxidants. We hypothize that using α-tocopherol and ascorbic acid as adjunct therapy of severe acute lower respiratory infection (ALRI) in infants may have a beneficial impact on the disease severity and recovery by reducing oxidative stress.
The lung is exposed to oxygen and contains unsaturated fatty acids that are substrates for lipid peroxidation. Therefore, the lung is thought to be at a higher risk of reactive oxygen species (ROS)-mediated injury. The pharmacologic interventions to prevent this phenomenon may be mediated through downregulation of proinflammatory cytokines, blocking of neutrophil infiltration, or inhibiting ROS formation. Inhaled bacterial endotoxin (lipopolysaccharide) is known to evoke acute airway inflammation (Rylander et al., 1983); in this process, alveolar macrophages promote the production of inflammatory mediators and recruit neutrophils to produce many ROS that are thought to kill bacteria and other microorganisms. On the other hand, excessive production of ROS may cause acute tissue injury and organ failure. The antioxidants therefore may play both a harmful and a beneficial role. α-tocopheral is present in all cell membranes at low concentrations. It has cytoprotective properties that are mediated by their ability to scavange ROS and thus prevent lipid peroxidation of cell membranes (Chow et al., 1991; Traber et al., 1995). Treatment with high doses of α-tocopherol has shown benefit in animal models of acute respiratory distress syndrome (Demling et al., 1995; Suntres et al., 1996; Fan et al., 2000; Rocksen et al., 2003). In an animal model, α-tocopherol was shown to prevent lung injury in endotoxin-induced airway inflammation (Rocksen et al., 2003). In a randomized double-blind trial, a modest dose of Vitamin C (200 mg daily) rendered clinical benefit in hospitalized elderly patients with acute respiratory infection (Hunt et al., 1994). Vitamin C supplementation was also shown to benefit subjects doing heavy physical exercise, in reducing the incidence of upper-respiratory infection (Hemila et al., 1999).
In a randomized double-blind placebo-controlled trial, we evaluated the role of two nutrients, α-tocopherol and ascorbic acid as adjunct therapy of pneumonia in infants and young children. In this study, we also evaluated the effect of these two nutrients on oxidative stress, a putative mechanism for severe disease due to pneumonia in infants and young children, and on cell-mediated immune status.
Patients and methods
Children aged 2–35 months of either sex who were sufficiently ill to be admitted into the inpatients services of BC Roy Memorial Hospital for Children, Kolkata, with a clinical diagnosis of ALRI were considered for inclusion in this study. This is a charitable hospital run by the Government and treatment is provided free. In the event of any shortage of medicines needed for patient care the research project fund provided them. Owing to extreme bed constraint only those children who appear very ill to the pediatrician are admitted to the inpatient services. Age group selected was based on the reported peak incidence of acute lower respiratory infection in children (Stansfield et al., 1993). The criteria for inclusion in the study were similar to those described in an earlier study (Mahalanabis et al., 2004) and was briefly stated. A diagnosis of ALRI was made by the presence of cough and fast breathing (respiratory rate >50/min for 2–11 months and >40/min for 12–35 months of age) or lower-chest indrawing (Stansfield et al., 1993), and of severe ALRI when ALRI was associated with either (i) cough combined with crepitation or bronchial breathing on auscultation, or (ii) one of the following severity indicators: not able to drink or feed, marked lethargy or irritability, nasal flare, and drowsiness. A child with severe ALRI as defined above was included into the study. Children with obvious marasmus or oedema, or with nonrespiratory severe infection such as meningitis or bloody diarrhoea, or congenital heart disease, or another gross congenital malformation were excluded. A child having rash with fever was also excluded. Once the patient was found eligible, informed consent was obtained and the child assigned to the study. The study protocol was approved by the Ethical Review Committee of the Society for Applied Studies. Patient recruitment took place from August 1998 to February 2001.
A master randomization schedule to arrange treatment assignment was prepared by a person not associated with the study, using permuted blocks of random numbers. The medicine bottles (separate for α-tocopherol and ascorbic acid) and identical placebo in taste consistency and colour were prepared by a pharmaceutical manufacturer and was supervised by a qualified pharmaceutical chemist acting as a consultant on our behalf. Random samples of the bottle mixture of α-tocopherol were tested by high-performance liquid chromatography for α-tocopherol. On the basis of master randomization chart, randomization was incorporated in the serially numbered bottles containing medicines or placebo, by a person not involved in the study. The bottle serial number corresponded with the patient's serial number. As an example, if the patient no. 6 falls in the intervention group, the two bottles, one labelled vitamin E and one labelled vitamin C, with serial no. 6 on each will contain α-tocopherol and ascorbic acid, respectively. Likewise, if patient no. 8 falls in the placebo group, both the bottles with serial no. 8 will contain placebo for α-tocopherol and ascorbic acid.
All patients received a standard schedule of treatment for pneumonia and associated problems, based on the existing practice of the hospital, which included antibiotics, bronchodilators and oxygen as required. On the basis of current practice in this hospital, the following antibiotic regime was used. All study children were treated with a combination of cloxacilin and gentamycin parenterally as a first-line antibiotics. If there was no improvement in 48 h or deterioration during the course of treatment, the physicians took a clinical decision to change the antibiotic regime to cefotaxime or ceftriaxone parenterally. Patients received α-tocopherol 200 mg and ascorbic acid 100 mg twice daily or placebo for 5 days.
According to the consensus among the paediatricians of this hospital, 60% of the patients would have achieved ‘clinically cured’ or ‘much improved’ status after 5 days. After treatment with α-tocopherol and ascorbic acid for 5 days as adjunct therapy, we assumed 80% of them would have achieved this status, as judged by the clinician. This degree of improvement was felt to be worthwhile for such an intervention. The calculated number in each group (with 80% power and 5% significance level) would be 83 children, including 5% with anticipated withdrawal. By using similar assumptions for the proportion of patients in whom tachypnoea or fever would have resolved after 5 days of treatment, the calculated sample sizes would be similar.
Serum α-tocopherol levels were measured using HPLC, at admission and discharge. Thiobarbituric acid reacting substances (TBARS) was measured by methods described earlier (Khaled, 1994). The predominant end product of all lipid-peroxidation activity is malondialdehyde (MDA), which reacts with thiobarbituric acid (TBA) by giving a pink colour detectable spectrophotometrically between 532 and 535 nM (Draper et al., 1993). MDA and similar products react with TBA, giving a complex of TBA reacting substances (TBARS). TBARS gives a measure of lipid peroxidation. These two blood tests were performed in a subsample in whom adequate blood samples could be obtained.
In addition to documentation of standard clinical features, the pediatricians recorded their clinical judgement as to whether a child has attained a clinically ‘cured’ or ‘much improved’ status based on the following criteria: (a) alertness and general well being, (b) resolution of respiratory distress (i.e. tachypnoea and/or lower-chest indrawing), (c) how well the infant feeds, and (d) resolution of fever. Major outcome variable was the time taken for this composite illness indicator to resolve. Clinical features were evaluated by the three study pediatricians (MB, DP, and SG) unaware of treatment allocation, and were recorded twice daily in the morning and evening. Evaluation was made by two or more pediatricians at a time and a consensus opinion was recorded. For clinically ‘cured’ status patients met all four, and three of the four criteria for ‘much improved’ status. Additional outcome variables examined were the time for the resolution of tachypnoea, fever, and feeding difficulty. We also evaluated the effect on oxidative stress as indicated by TBARS and on cell-mediated immunity indicated by delayed type hypersensitivity response to multiple skin antigens.
We evaluated cell-mediated immunity (as indicated by delayed hypersensitivity) by using Multitest CMI® (Pasteur Merieux Serums & Vaccines, Lyon, France). Multitest CMI® is a disposable plastic applicator consisting of eight sterile heads, each with multiple prongs preloaded with seven delayed hypersensitivity skin test antigens and a glycerin negative control. It is applied on skin intradermally. An induration of 1.5 mm or more, after 48 h is taken as positive response to the antigen. The antigens used are tetanus, diphtheria, streptococcus (group c), tuberculin (old), candida (albicans), trichophyton (mentagrophytes), and proteus (mirabilis). The control head contains glycerin. One research assistant not involved in the conduct of this study was trained to administer and read the results of the multitest CMI.
Data were edited using Epi Info version 6.03 software (CDC, Atlanta, USA and WHO, Geneva). Major interest of the study was to evaluate the effect of α-tochopherol and ascorbic acid on the clinical course of illness due to severe ALRI in infants and young children. We therefore used survival (time to an event) analysis techniques to compare the duration of the illness indicators, which permit us adjust for censored or truncated data. Clinical illness indicators used were time to resolution of (a) a very ill clinical status as judged by the paediatrician, (b) fever, (c) feeding difficulty or inability to feed, and (d) tachypnoea. Prognostic factors like age and sex were adjusted for, using Cox proportional hazards regression analysis. The hazards ratio indicates the ratio of recovery rates from the illness indicators in supplemented to the unsupplemented groups at any point in time during the study and values greater than 1 are in favour of α-tocopherol- and ascorbic-acid supplemented group. The mean duration of illness indicators were obtained from the Kaplan–Meir product-limit estimate of the survivor function, and the standard error of the mean was calculated by the method of Klein and Maeschberger (Klein et al., 1997). A software Stata release 7.0 (Stata Corporation, Texas, USA) was used for survival analysis.
In total, 174 children (Figure 1) were enrolled and nine children died during the study, six in the control group and three in the intervention group (rate ratio=0.48, 95% CI: 0.12–1.85, P=0.32, Fisher exact two-tailed). The three in the study group died at 10, 33, and 77 h and the six in the control group died at 9, 12, 20, 33, 38, and 118 h, after admission. Two children in the control group were withdrawn from the study by their parents, one at 24 h, and the other at 44 h. One each in control and intervention groups were diagnosed to have congenital heart disease at 19 and 43 h of study, respectively, and were removed from the study ward. One in the intervention group developed measles at 18 h and was transferred to another hospital. No secondary case of measles occurred in the ward following admission of the child with measles. Only two children were admitted to the study 5 days before and no child was admitted to the study during 10 days following the admission of the child with measles. For any child who was removed from the study ward, died, or taken away by parents, data up to the point of removal were included in the analysis. Therefore, analysis was performed on the basis of ‘intention to treat’. Immunization status was similar in the two groups. In the control and intervention groups, respectively, 43 and 48% received three or more doses of oral polio vaccine, 24 and 18% received measles vaccine, 80 and 82% received BCG vaccine, and 40% in each received at least three doses of DPT vaccine. The apparently low immunization rate is largely owing to young age of the patients. National immunization data are reported for 1-year-old children. The treatment groups were broadly similar at baseline (Table 1). Patients came from poor families. Male preponderance reflected the pattern of admission in this hospital. More than 70% were infants under 1 year. The rate of breastfeeding was high. A high proportion received antibiotics before visiting the hospital.
In the control group, 79% and in the study group, 78.7% attained much improved or cured status at discharge (rate ratio=1.01, 95% CI: 0.86–1.18, P=0.95). The mean and standard error of the duration of illness indicators, based on survival analysis for censored data were broadly similar in the two groups (data not shown). The recovery rate ratios based on Cox proportional hazards models with duration of illness indicators (both primary and secondary outcome measures) as dependant variables do not indicate any benefit from the adjunct therapy of pneumonia in infants, with ascorbic acid and α-tocopherol (Table 2). We should however note that the sample size was calculated for the primary outcome measure only. Results on the secondary outcome measures lend support to the primary outcome. Rate of change in the antibiotic regime in the two groups was similar (31.5% in control and 32.9% in the intervention group). Trials of vitamin E and or C in childhood pneumonia or ALRI is lacking in the literature. Bacterial pneumonia such as pneumonia due to pneumococcus or Haemophilus influenzae are common causes of mortality and morbidity in developing countries. One can speculate that natural antioxidants like vitamin E and C may be more effective in ALRI of viral aetiology. The effect of α-tochopherol and ascorbic acid supplementation on oxidative stress in these infants was evaluated by measuring the thiobarbituric acid-reacting substances (TBARS) in the plasma. TBARS concentrations on admission, at discharge, and on follow-up after 2 weeks showed no significant difference between the two groups (Table 3). The values were generally high at admission, discharge, and follow-up. Serum α-tocopherol levels at admission were similar in the two groups and they were generally low. In control group, 24 out of 56 (42.9%)and in the intervention group, 33 out of 62 (53.2%) had low levels (<8.8 μmol/l), and at discharge, 35.1% in control group and 16.9% in the intervention group had low α-tocopherol levels (P=0.021). At discharge, its concentration rose in the study group and the difference with the control group was significant (P=0.002). At 2 weeks of follow-up, the serum α-tocopherol levels were similar in the two groups. Serum α-tocopherol values therefore improved on daily supplementation during acute lower respiratory infection; which came down on stopping the supplement.
Cell-mediated immunity was measured by delayed type skin response to intradermal multiple antigens at admission, and at 2 weeks follow-up. At admission two out of 57 tested in the control group were positive for one and two antigens, respectively, and two out of 54 in the treatment group were positive for two and four antigens, respectively. At 2 weeks after admission: four out of 27 tested in the control group were positive, that is, one for four, two for two each and one for one antigen; and two out of 27 in the treatment group were positive for four antigens each. Cell-mediated immunity therefore was poor in both groups at admission and at follow-up, after 2 weeks of admission. It may be noted that a large number of the study children were immunized with DPT and BCG, which contained three of the seven antigens used for the intradermal test. In all, 51% received at least two doses and 75% received at least one dose of DPT immunization. Kaplan–Meier survival curves for the duration of illness indicators comparing the treatment group with the control group (Figure 2) further illustrate that the recovery from illness indicators was similar in the two groups.
In this study, vitamin E and C treatment of infants and young children hospitalized with pneumonia did not render any worthwhile benefit for the course of clinical illness. Furthermore, although the values of TBARS, an indicator of oxidative stress, were high in these very sick infants, administration of two antioxidant vitamins did not beneficially influence the degree of oxidative stress. These infants were generally very anergic during the acute phase of illness as evidenced by a lack of response to intradermal administration of multiple antigens. As stated earlier, three of these seven antigens that is, diphtheria, tetanus, and tuberculin were administered to a large majority of these infants by way of routine immunization. Immune response to these antigens following administration of vitamin E and C was also very poor. At 2 weeks follow-up, the state of anergy remained high. One explanation for this high degree of poor cell-mediated immune response in these infants may be due to the fact that majority of them may have had a viral infection with or without a bacterial infection, which is known to cause depression of cell-mediated immunity.
Oxidative stress occurs when there is an imbalance between oxidants and antioxidants in a living organism. Living cells are exposed to oxidants arising from external and internal sources. External sources include air pollutants, harmful gases (e.g., ozone), ionizing and nonionizing radiations, chemicals and toxins, and pathogenic bacteria and viruses (Menzal, 1994; Podda et al., 1998; Halliwell et al., 2000). Many childhood diseases have been linked with oxidative damage and has recently been reviewed (Granot et al., 2004). Current interest in oxidative stress and antioxidants arise from the putative role of ROS in many diverse conditions such as ageing, atherosclerosis, cancer, inflammatory bowel diseases and intestinal reperfusion injury, neurodegenerative disorders, chronic renal failure, viral infections, and the immune response (Jacques et al., 1988; Harats et al., 1990; Harris et al., 1992; Powell et al., 1994; Haklar et al., 1995; Schwartz et al., 1996; Kohen et al., 1997; Stone, 1997). As we stated earlier, high doses of α-tocopherol showed benefit in animal models of respiratory distress syndrome and endotoxin-induced air way inflammation (Demling et al., 1995; Suntres et al., 1996; Fan et al., 2000; Rocksen et al., 2003). In a randomized double-blind placebo-controlled study, natural antioxidants including vitamins E and C improved lung function and reduced airway inflammation in heaves-affected horses (Kirschvink et al., 2002). Inflammatory processes are associated with the release of ROS that originate from the respiratory burst in activated neutrophils. Hydroxyl radicals mediate oxidative damage and a reaction between superoxide anions and nitric oxide (NO), and the presence of iron contribute to their production (Beckman et al., 1990). Enhanced oxidative stress in cystic fibrosis patients is believed to be a consequence of both increased production of ROS during infections and inadequate antioxidant defences due to malabsorption of fat soluble vitamins (Portal et al., 1995).
Healthy breast-fed infants were shown to have two-folds higher plasma TBARS values than that of formula-fed infants and this was attributed to a high content of long chain polyunsaturated fatty acids (PUFA) of breast milk (Granot et al., 1999). PUFAs are a major substrate of lipid peroxidation. We may note that overwhelming majority of the infants in the present study were breast-fed; which may explain the high TBARS values in them. Spitzer (1995) suggested that the presence of lipids susceptible to oxidation by scavenging oxygen radicals may contribute to cellular-antioxidant protection in the early stages of development when other antioxidant defence mechanisms may be less effective. Furthermore, a beneficial role for oxidative stress has been suggested in many diverse processes in nature including cell proliferation, gene activation, and expression and activation of spermatozoa (De Lamirande et al., 1995; Remacle et al., 1995). We therefore need a better understanding of oxidative processes in a growing infant; that will help us better design interventions with antioxidant therapy and determine optimum nutritional antioxidant supplementation in infancy and childhood.
To conclude, although the oxidative stress was found to be high in these infants with severe ALRI, two antioxidant nutrients failed to beneficially influence their course of illness. The reduced cell-mediated immune response in them suggests that severe ALRI in infants may cause immune dysfunction.
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The study was supported by a research Grant from Nestle Foundation, Switzerland. We thank Dr Mrinal Kanti Chatterjee (Superintendent), the doctors and healthcare workers of BC Roy Memorial Hospital for Children and ML Chakrabarti of Kothari Medical Research Centre, Kolkata, for their assistance in conduct of the study. We thank Mr Jakir Hossain for computer data management and analysis, and Mr Subodh Karmakar for typing and editing the manuscript.
Guarantor: D Mahalanabis.
Contributors: DM was responsible for study design, data analysis and writing the manuscript. MB, DP and SG took part in case management and evaluation, skin antigen tests and data collection, and assisted in writing the manuscript. SS and MAW took part in laboratory tests and interpretation and assisted in the writing of the manuscript. MAK took part in the study design, in establishing and standardizing TBARS procedures and interpreting the results, and in writing the manuscript.
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