The Journal of International Medical Research 1996; 24: 325 - 330

Essential Fatty Acids: the Effects of Dietary Supplementation among Children with Recurrent Respiratory Infections A

VENUTA\

C SPANii,

L LAUDIZI\

F

BETTELLI\

A

BEVERELLi AND

ETuRCHETTd

'Department of Gynaecology, Obstetrics and Paediatrics, University of Modena, Modena, Italy; 2Nutrition Research Centre, Department of Biochemistry 'G. Moruzzi', University of Bologna, Bologna, Italy

The impact of dietary supplementation with essential fatty acids (EFA) on recurrent respiratory infections (RRI) in children was evaluated by means of a randomized crossover double-blind study. Linoleic acid (596 mg/day) and alpha-linolenic acid (855 mg/day) as a commercial preparation or placebo (olive oil) were administered for two consecutive winter seasons (November to February, To T 12o) to 20 children affected by RRI, aged between 36 and 49 months. Plasma levels of n-3 and n-6 metabolites increased after the administration of EFA. The number of infective episodes, days' fever and days' absence from school were reduced significantly during the observation period (extended from T 120 to T18o) in children receiving EFA supplementation. Our results suggest that n-3 and n-6 polyunsaturated fatty acids may play a favourable role in the defence mechanism ofthese subjects. KEY WORDS:

ESSENTIAL FATTY ACIDS;

INFECTION; IMMUNE SYSTEM; CHILDREN

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RECURRENT RESPIRATORY

INTRODUCTION us by their family doctor (paediatrician) for RRI. Inclusion criteria were: attendance at day care schools, a history of RRI with at least 10 episodes in the preceding 12 months and absence of recognized immunodeficiency. They were aged between 36 and 49 months at the beginning ofthe study. A written, informed consent was obtained from the parents. The study design was a double-blind cross-over with placebo control. Essential fatty acids were administered in the form of a commercial product containing (i)"-6 as linoleic acid and (i)"-3 as alpha-linolenic acid, derived from linseed and soya oil in a ratio of 1:1.4. The dose administered was 2.25 ml/day, containing respectively alpha-linolenic acid 855 mg and linolenic acid 596 mg. Each child was randomized to the verum or placebo group and received either the alimentary supplement or the placebo (olive oil) in equal quantities for four months (November to February). The following winter the group that had received EFA was given placebo and vice versa for another four months. The observation period included the duration of administration of EFA or placebo (To - T 12o). This was extended to the two months following termination of treatment

Each day, paediatricians encounter the problem of recurrent respiratory infections (RRI). Although this pathology has a favourable evolution with time, it often places considerable stress on the family and has a substantial impact on the health service. Early socialization, weather conditions, the domestic microecology, urban pollution and socioeconomic status are implicated in a complex and multifactorial epidemiology. Despite clear evidence of various humoral, cellular and granulocyte deficits from studies of the immune system of these children, no single cause has been identified. 1 - 2 Essential fatty acids (EFA) have been used as dietary supplements in infant formula feeds and in the treatment of atopic eczema in paediatrics. Their biological role has also been evaluated in premature infants, children with cystic fibrosis, hyperkinetic children and subjects with food allergies. The increasing number of recent studies of EFA demonstrates a growing interest and breadth of areas for use in the developing

child." During outpatient follow-up of children with atopic eczema who were given EFA, parents noted and reported not only an improvement in cutaneous symptomatology but also a reduction in the incidence of the respiratory infections to which these children are normally more susceptible. This observation appeared worthy of further study and it was therefore decided to evaluate the clinical impact of alimentary supplementation with EFA on children affected by RRI.

(T , 2o - T , 8o).

A diary was provided for parents to record the type and duration of the infective episode, kind of therapy administered and number of days missed from school. Fever was chosen as the parameter for duration of respiratory infection because of objectivity, as other symptoms, such as sore throat or earache, were more subjective and could not easily be described by a very young child. Other objective signs such as a runny nose or cough tend to persist in these children for the entire winter season and thus are not

PATIENTS AND METHODS Thirty-eight children of both sexes .were included in the study. They were referred to

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appropriate parameters for a comparative evaluation. No pharmacological modulators of the biological response were permitted during the study. Possible immunodeficit carriers among all the children in the trial were identified by studying the lymphocyte phenotype for the antigens CD3 (T cells), CD4 (helper T lymphocytes) and CD8 (cytotoxic T lymphocytes) by direct immunofluorescent technique (cytofluorimeter FACSAN and monoclonal antibodies, Becton Dickinson) and the levels of circulating immunoglobulins, including the subclasses IgG (nephelometric method, Behring Nephelometric Analyser, with monospecific antisera, The Binding Site, Birmingham, UK).

RESULTS Four children were excluded at the beginning of the study because of an immunoglobulin deficit, three for partial IgA deficit, one for a deficit of the subclasses IgGz (below two SD). The lymphocyte phenotypes CD3, CD4 and CD8 of the subjects studied did not differ significantly from a paediatric population of 42 subjects of the same age group used as a control. Fourteen children were excluded because of protocol violations. Only one child dropped out of the study because of secondary effects attributable to the dietary supplementation. The loose stools and abdominal pain experienced by some children with the administration of the EFA at the prescribed dosage disappeared when the dosage was halved. The remaining 20 children completed the study.

PLASMA ANALYSIS At the beginning and end of the two treatment periods the plasma levels of linoleic acid, alpha-linolenic acid and their metabolites were also determined. The modified Folch method was adopted for the extraction of the plasma lipids and, once extracted, the methyl esters of the fatty acids were obtained by the Stoffel method. Gas chromatographic analysis was performed on the methyl esters using a Carlo Erba Instrumentation HRGC 5160 (Carlo Erba, Rodano, Milano, Italy). This device was equipped with a fused silica capillary column Supelco SP2340 (Supelco, Bellefonte, California, USA), 30 m long, with a 0.32 mm internal diameter and film thickness of 0.2 /-lm. The analysis temperature began at 160 DC and reached 210 DC with an increase of 8 DC/min. The carrier gas used was helium with a flow of 2 mllmin in the column. The peaks were integrated with a Carlo Erba DP 700 integrator and the results were expressed as mol % of fatty acids. STATISTICAL ANALYSIS The results were evaluated Student's r-test for paired data.

with

DETERMINATION OF THE PLASMA POLYUNSATURATED FATTY ACIDS The levels of polyunsaturated fatty acids of the n-6 and n-3 series in the plasma after dietary supplementation with linoleic and alpha-linolenic acid are shown in Table 1. Greater increases in the n-3 fatty acids, significant for all the metabolites, were found. This is due to the higher n-3 content of the dietary supplement administered. On the whole, however, n-6 increased, with a significant increase of arachidonic (22:4 n-B] and docosapentaesanoic (22:5 n-B] acids, which represents an increased activity of the enzymes involved in the lengthening and synthesis of the carbon chain (A-desaturase and elongase). The plasma levels of polyunsaturated fatty acids before and after the administration of olive oil are shown in Table 2. The only detectable datum is a modest and not statistically significant increase of n-B,

the

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TABLE 1 Levels of n-6 and n-3 fatty acids before and after dietary supplementation with linoleic acid and alpha-linolenic acid (mols %)

To

T120

Statistical significance*

18:2 n6 18:3 n6 20:3 n6 20:4 n6 22:4 n6 22:5 n6 total n-s

23.17±5.31 0.35 ± 0.24 1.85 ± 0.50 6.05 ± 1.6 0.32 ± 0.10 0.23 ±0.15 31.97

22.93 ± 3.39 0.38 ± 0.17 1.93 ± 0.45 6.84 ± 1.33 0.37±0.14 0.42±0.14 32.87

NS NS NS

18:3 n3 20:5 n3 22:5 n3 22:6 n3 total n-s

0.28 ± 0.17 0.46 ± 0.26 0.32 ± 0.11 1.32 ± 0.61 2.38

0.69 ± 0.48 0.66 ± 0.33 0.52±0.12 2.03 ± 0.72 3.90

p= 0.0327

NS p= 0.001

p= 0.0017 p= 0.0405 p= 0.0002 p= 0.0011

'Student's t-test for paired data.

TABLE 2 Levels of n-6 and n-3 fatty acids before and after supplementation with olive oil (mots %)

To

7120

Statistical significance*

18:2 n6 18:3 n6 20:3 n6 20:4 n6 22:4 n6 22:5 n6 total n-s

22.91 ±4.57 0.34 ± 0.20 1.78 ± 0.55 5.88 ± 1.44 0.34 ± 0.18 0.25 ± 0.14 31.50

23.01 ± 5.33 0.37 ± 0.22 1.62 ± 0.41 6.13 ± 1.69 0.33 ± 0.18 0.28 ± 0.17 31.74

NS NS NS NS NS NS

18:3 n3 20:5 n3 22:5 n3 22:6 n3 total n-3

0.33 ± 0.16 0.39 ± 0.20 0.33 ± 0.13 1.35 ± 0.77 2.40

0.29 ± 0.17 0.4 ± 0.18 0.28 ± 0.12 1.38 ± 0.52 2.36

NS NS NS NS

'Student's t-test for paired data.

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TABLE 3 Effect of the dietary supplementation with n-3 and n-6 on the number and duration of recurrent respiratory infections

No. of infective episodes/month Days of fever/month Days missed from school/month

Placebo

Supplement

(To - T,2o)

(To - T,80)

1.39 4.15 6.94

± 0.75 ± 0.98 ± 1.82

0.86 2.98 5.17

± 0.58 ± 1.56 ± 1.49

Statistical significance * p= 0.019 p= 0.007 p= 0.002

'Student's t-test for paireddata.

probably due to the linoleic acid content of the olive oil.

based on the observations made by the parents of children with atopic eczema. A greater deformability and elasticity is given to the cellular membranes by the presence of double bonds in long carbon chains. The improved intracellular cohesion that results can cause a barrier effect which is particularly important at the respiratory mucosal level. The n-6 fatty acids are precursors of the prostaglandin (PG) molecules of the PGE, - PGEz series; the n-3 acids are precursors of PGE3 • The modulation of the inflammatory response is determined by the equilibrium between the different PGs. Some n-3 and n-6 polyunsaturated fatty acids interfere with the function of leucocytes with non-eicosanoid mechanisms. The EFAs and their metabolites can alter many leucocyte functions: the respiratory burst (which has been studied in vitro), chemiluminescence superoxide anion production, migration and chemotaxis of neutrophils."" The EFAs and their metabolites are indispensable to the antiviral activity of interferon. In addition, the viruses, as part of their attack strategy, reduce the capacity of the cell to produce 6-desaturated AGE.8 ,g The idea that these molecules have a very important role in the regulation of the immune system is supported by the global

CLINICAL EFFECT OF EFA SUPPLEMENTATION ON RRI FREQUENCY Table 3 contains the number of infective episodes per month, days of fever per month and days missed from school per month counted during the observation period (To T '80) with EFA and with placebo. The results demonstrate a significant reduction in the number and duration of episodes of RRI with the dietary supplementation of n-3/n-6 as compared with placebo. The adoption of a cross-over design provides an internal comparison for every child and minimizes the 'improvement with age' effect typically seen with RRI and the variations in morbidity from year to year.

DISCUSSION Prolonged dietary supplementation with polyunsaturated fatty acids of the n-3 and n-6 series results in considerable variations in the metabolic patterns of these substances. In children with frequent RRI this seems to correspond to a reduced incidence in the number and duration of infective episodes,

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consideration of these data. The favourable effect that we have detected in the frequency of RRI in children with atopic eczema can be placed in this wider context, and it is our opinion that the immunological significance

of a diet supplemented with EFA merits further evaluation. The confirmation of our results in a much larger paediatric population could pave the way for use of EFAs as biological response modulators.

REFERENCES 1 Herrod HG, Blaiss MS, Valen ski WR, Gross S: Cell-mediated immune status of children with recurrent infection. J Pediatr 1995; 126: 530 - 536. 2 Gross S, Blaiss MS, Herrod HG. Role of immunoglobulin subclasses and specific antibody determinations in the evaluation of recurrent infection in children. J Pediatr 1992; 121: 516 - 522. 3 Bottai F, Franchini F, Cocchi C, et a1: Recenti acquisizioni sull'uso degli acidi grassi essenziali in pediatria ai limiti tra dieta, preventione e terapie. Pediatr Med Chir 1994; 16: 109 -115. 4 Hardy SJ, Robinson BS, Poulos A, et a1: The neutrophil respiratory burst. Responses to fatty acids, N-formylmethionylleucylphenylalanine and phorbol ester suggest divergent signalling mechanisms. Eur J Biochein 1991; 198: 801 806. 5 Varming K, Schmidt EB, Svaneborg N et aJ: The effect of n-3 fatty acids on neutrophil chemiluminescence. Scand J CJin Lab Invest 1995; 55: 47 - 52. 6 Badwey JA, Curnutte JT, Robinson JM et

aJ: Effects of free fatty acids on release of superoxide and on change of shape by human neutrophils. J BioJ Chern 1984; 59: 7870 - 7877. 7 Ferrante A, Goh D, Harvey DP et a1: Neutrophil migration inhibitory properties of polyunsaturated fatty acids. J Clin Invest 1994; 93: 1063 -1070. 8 Hanigan GE, Williams BR: Signal transduction by interferon-alpha through arachidonic acid metabolism. Science 1991; 251: 204 - 207. 9 Horrobin DF: Postviral fatigue syndrome, viral infections in atopic eczema, and essential· fatty acids. Med Hypotheses 1990; 32: 211- 217.

A Venuta, C Spano, L Laudizi, F Bettelli, A Beverelli and E Turchetto Essential Fatty Acids: the Effects of Dietary Supplementation among Children with Recurrent Respiratory Infections The Journal of International Medical Research 1996; 24: 325 - 330 Received for publication 23 February 1996 Accepted 5 March 1996 © Copyright 1996 Cambridge Medical Publications

Address for correspondence DRANDREA VENUTA

Dipartimento di Scienze Ginecologiche Ostetriche Pediatriche, Universita di Modena, via del Pozzo 41100, Modena, Italy.

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