The achievement of normal growth and adequate nutritional status in patients with cystic fibrosis (CF) is a major clinical issue (1). Malnutrition and growth retardation negatively affect prognosis and pulmonary function (2). A free and balanced diet, supplying 120% to 150% of the recommended energy dietary allowances for age, should be prescribed, starting from the time of diagnosis (3,4). This approach should allow CF patients to achieve their genetic growth potential by approaching an optimal nutrition status (5).
The efficacy of this nutritional strategy is indicated by the significant improvement of weight and height percentiles documented during the last 2 decades for the CF population (6), together with a better survival. In addition, nutritional intervention in infancy has been shown to have long-term growth advantages (7). These observations have led to a greater attention to the nutritional status of CF patients; however, selective nutritional deficiencies are emerging as significant clinical problems (8,9).
Essential fatty acid (EFA) deficiency, mainly involving the parental n-6 compound, linoleic acid (LA; C18:2n-6), is a common finding in CF, but also long-chain polyunsaturated fatty acids (LCPUFA), particularly docosahexaenoic acid (DHA; C22:6n-3), the most active n-3 derivative, is present in very low concentrations (10,11). In contrast, the levels of arachidonic acid (ARA), LCPUFA derived from LA, are generally preserved or even increased, thus raising the hypothesis of a pathological regulation of the release of ARA in CF (12). The resulting defect in the regulation of EFA metabolism could be related to the functional defect of the cystic fibrosis transmembrane regulator (CFTR) protein (13,14). In the CFTR knockout mice, a similar fatty acid imbalance in the membranes of organs where CFTR is mainly expressed (pancreas, lung) was described by Freedman et al. (15), and the oral supplementation with DHA corrected this imbalance while reversing the typical lesions at the pancreatic and pulmonary level. More recently, alterations of the fatty acid pattern similar to those observed in the CFTR knockout mice were observed by the same group of investigators in the nasal and rectal mucosa of CF patients (16). However, a study carried out in 2 different CF mouse models could not confirm these findings, thus suggesting that the membrane EFA imbalance is not an intrinsic characteristic of the CF genotype and that the altered EFA composition may be a secondary phenomenon, possibly related to inflammation or malnutrition (17).
To test the hypothesis that a poor fatty acid status is related to the clinical conditions and/or fatty acid intake rather than being an intrinsic characteristic of CF, we have evaluated dietary intakes and the fatty acid profile of plasma phospholipids (PLs) as an index of the EFA status in CF patients and in a reference group of healthy children (18). In CF patients, both parameters were then correlated with relevant clinical outcomes such as growth, pulmonary function and the Shwachman score.
PATIENTS AND METHODS
The study was carried out from February to June 2003. Of the 450 patients followed at the CF Center of the University of Milan, all CF patients in the primary school-age range consecutively seen at the CF Center of the University of Milan in that period were considered for enrolment. Criteria for inclusion were also good family compliance with the visits and the medical advice (inclusive of drug therapy and diet) and stable conditions at entry with no evidence of pulmonary exacerbation.
Patients had been diagnosed on the basis of increased sweat chloride concentrations on at least 2 occasions (Gibson and Cook's method) (19) and/or the presence of 2 mutations of the CFTR gene associated with the disease. An extensive molecular analysis had been performed by denaturing high-performance liquid chromatography (20) whenever direct analysis of the 31 most common CFTR mutations (by means of PCR/OLA Cystic Fibrosis Assay) had not permitted a definition of CFTR genotype. CFTR genotype was severe in 30 patients, mild in 2 and remained undefined in 5, 3 of whom had pancreatic insufficiency and 2 had pancreatic sufficiency. Pancreatic insufficiency (present in 33 patients) was defined by means of faecal elastase as determined by immunoenzymatic method (21).
All the patients with pancreatic insufficiency were under treatment with pancreatic enzymes; doses were established on the basis of meal fat content, presence of clinical symptoms and sequential steatocrit determination (22). A high-fat, high-energy diet was prescribed to achieve a daily energy intake in the range of 120% to 150% of the recommended dietary allowances. At enrolment, all patients underwent clinical evaluation with anthropometric measurement and determination of the Shwachman score (23). Height and weight transformed in z scores were matched with reference Italian children using the CDC-WHO reference values (24) by means of the ANTHRO pediatric anthropometry software program (version 1.01, 1990, Centres for Disease Control and Prevention, Atlanta, GA).
Forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were determined by means of spirometry and expressed as percentage of predicted for age (25).
Blood samples were drawn after an overnight fast for the determination of plasma and PL fatty acid composition by capillary gas-liquid chromatography (26). At the same time, a survey on dietary habits was performed by giving the parents a specifically conceived questionnaire.
The reference group was composed of 68 healthy controls (40 boys and 28 girls; mean age, 8.0 ± 0.7 yrs; age range, 7–9 yrs), who had been followed up from birth at the Department of Pediatrics of the San Paolo Hospital of the University of Milan for the identification of early markers of acquired metabolic diseases (27–29) and had undergone a dietetic survey using the same questionnaire and plasma fatty acid determination in the same laboratory.
The study design has been approved by the Hospital Ethical Committee, and parents gave their informed consent at enrollment.
Assessment of Dietary Habits
A food-frequency questionnaire previously validated for the pediatric age was used (30). The questionnaire contains a list of 116 specific items, different for different ages, with a space for frequency (daily, weekly, bimonthly, monthly) and a standard size, thus allowing for quantitative analysis. Mothers were interviewed for approximately 50 minutes, and each meal was analysed to establish which food was eaten and how often it was eaten. Usual portion sizes were estimated using household measures, the weight of purchase (eg, pasta) or unit (eg, fruit juice). The following food items were considered for frequency of consumption and coded accordingly: rice, bread, pasta, olive oil, seed oil, butter, whole cow's milk, skimmed cow's milk, yogurt, cheese, eggs, red meat (beef), white meat (veal and poultry), fish, green vegetables, legumes, cakes and snacks. A 24-hour recall was further conducted at the end of the interview to standardize the usual serving size. Quantification and analysis of the energy intake and nutrient composition were performed with an ad hoc PC software program developed at our Department and based on the Food and Nutrient Data Base issued by the Italian Institute of Nutrition (31). The energy intake calculated for each subject was expressed as percentage of intake compared with the corresponding recommended dietary allowance for age and gender issued by the Italian Society of Nutrition (32).
Commercial energy supplements (eg, Resource Fruit, Nidex, Meritene, Duocal) constitute an important source of daily dietary intake in a significant proportion of CF patients. Macronutrient content of these formulations was calculated from data reported on label and/or packaging and added to the database of the questionnaire adapted for CF. Most products are based on proteins and carbohydrates, and the few supplying fat do not include LCPUFA.
Analysis of Plasma Fatty Acids
Peripheral venous blood, drawn in fasting conditions, was collected in a tube with anticoagulant (sodium citrate) and centrifuged at 4000 rpm for 10 minutes. Plasma obtained was kept at −20°C until analysed.
Total lipids were extracted from plasma according to Folch et al. (26) by the addition of water and chloroform-methanol (2:1) in the presence of butylated hydroxytoluene (5 μg/mL) as antioxidant. The fatty acid composition of plasma total lipids and plasma PLs were separated by thin-layer chromatography and then analysed with high-resolution capillary gas-chromatography, as previously detailed (33,34). Individual detector outputs were identified using pure reference compounds (Sigma, cod.189-19) and by mass spectrometry (Trio 1000, Fisons Instruments, Italy) and expressed as weight percentage of fatty acid methyl esters. Peak areas were calculated on a Chrom-Card software (Chromcard version 1.19) using heptadecanoic acid as internal standard.
In CF patients, the following parameters were considered for possible correlation with dietary intakes and plasma fatty acid profile:
- Growth: anthropometric parameters were evaluated at the time of assessment.
- Shwachman score (23), the most widely used clinical score to assess disease severity in CF (the higher, the better), and sweat chloride concentrations as an indirect biochemical marker of the severity of the basic defect.
- Pulmonary function tests (FEV1 and FVC).
The t test for independent variables was used to investigate the statistical significance of the differences between means in CF patients and controls for normally distributed data (after Kolmogorov-Smirnov test), whereas the nonparametric Mann-Whitney U test was used for not normally distributed data (35). Given the distribution of values in controls, it was estimated that the number of CF patients included would allow for the detection of a 10% difference in LA and a 15% difference in DHA between groups as statistically significant (P < 0.05) with 90% power of the test.
The following associations were then investigated using the Pearson correlation coefficient or non-parametric tests (Spearman rank correlation coefficient) according to the type of distribution:
- Dietary macronutrient (proteins, carbohydrates, fat families— saturated, monounsaturated and polyunsaturated) with levels of LA, eicosatrienoic acid (ETA, C20:3n-9, a marker of EFA deficiency), DHA, ARA, n-6, n-3 and total polyunsaturated FA in plasma total lipids and PL as dependent variables;
- Levels of LA, ETA, DHA, ARA, n-6, n-3 and total polyunsaturated FA in plasma total lipids and PL with functional correlates (height z score, weight z score, FEV1, FVC, sweat chloride and Schwachman score).
During the study period, 43 primary school–age patients were seen at the CF center. Thirty-seven patients (17 boys and 20 girls; mean age, 8.0 ± 2.9 yrs; median, 8.1; range, 3.4–12.3 yrs) were enrolled. Of these patients, 2 had liver disease, 2 had diabetes, and 8 were chronically colonized by Pseudomonas aeruginosa. Six patients were discharged, 4 of whom were discharged because of poor compliance and 2 were discharged because of pulmonary exacerbations.
In CF patients, weight and height z scores were −0.35 ± 1.16 and −0.28 ± 0.99, respectively, whereas the correspondent figures in the reference group were 0.83 ± 1.73 and 0.55 ± 1.11. As expected, the differences between the 2 groups were significant; however, in CF patients, the average weight and height z score were within 0.5 z difference compared with the mean (z = 0), the limit usually indicated as meaningful, thus excluding severe malnutrition and growth failure. In addition, in our CF patients, ideal weight for height average values were satisfactory (99.4 ± 11.1%), and only 3 patients showed values below 85%. All CF patients had mild to moderate pulmonary involvement (FEV1, 106 ± 22%; FVC, 102 ± 18%). In our reference group, 16 subjects (23% of the group) were overweight according to accepted international standards (32), reflecting the present trend among young Western populations.
For each patient, the questionnaire was completed and the results are shown in Table 1. Total energy intake was significantly higher in CF patients than controls also when adjusted for body weight and the Italian Recommended Dietary Allowances adjusted for age and gender. The increase in energy intake occurred as a result of the relative increase in lipid intake (P < 0.0001); the relative carbohydrate intake was significantly lower (P < 0.0001) than controls, whereas the protein intake was comparable. Extra meals and energy supplements had been prescribed in 16 patients.
In CF patients, the percentages of energy derived from saturated and monounsaturated fatty acids were significantly higher compared with controls (12.9 ± 3.1% vs 9.8 ± 1.6%, P < 0.0001 and 11.2 ± 2.3% vs 10.3 ± 1.5%, P = 0.01), whereas the percentage of energy derived from polyunsaturated fatty acids (PUFA) was slightly lower (3.9 ± 1.0% vs 4.3 ± 1.2%, P = 0.05).
The fatty acid profile of plasma PL is shown in Table 2. Significantly higher percentage levels of saturated and monounsaturated fatty acids were found in CF patients compared with controls, whereas PUFA were significantly lower considering either the n-6 series (in both plasma total lipids and plasma PL) and the n-3 series (in plasma PL). Plasma n-6 LCPUFA were significantly higher in CF patients, whereas the n-3 LCPUFA series showed higher levels in plasma PL. There were no significant differences in plasma percentage levels of ARA, whereas eicosapentaenoic acid (the metabolic precursor of DHA) was higher in CF patients. Both LA and DHA were significantly lower in CF patients who also showed significantly higher levels of ETA.
With regard to the correlations between fat intakes and plasma fatty acids, no significant associations were found either in CF and control subjects.
We finally explored the correlations between dietary intakes and the PL polyunsaturated fatty acid percentage levels and clinical outcomes. The Shwachman score was negatively associated with long-chain n-6 PUFA and with ARA alone (r = 0.35, P = 0.03, and r = 0.32, P = 0.05, respectively; that is, the worst clinical condition is associated with higher n-6 LCPUFA and ARA levels). No associations were found between PL fatty acids and either anthropometric or lung function measures.
The purpose of this report was to test the hypothesis that a poor PUFA and/or long-chain PUFA status is related to the fatty acid intake and/or the clinical conditions, rather than intrinsic characteristics, of CF. We, therefore, have to assess the relationship between dietary intakes, plasma fatty acid profile and clinical parameters in children with CF in comparison to healthy controls.
As a first step, we have assessed the dietary intakes in a population of CF patients regularly followed up at our CF Center. To this aim, we have used a food-frequency questionnaire that, in other nutritional studies (27–29,36,37), permitted us to correlate early adiposity rebound (27,36), plasma fatty acids (28) and overall glycemic index (29,37) with dietary macronutrient intakes (protein, animal foods, and carbohydrate, respectively) from early infancy through later childhood. By using the same questionnaire in the present investigation, we could examine for the first time the plasma fatty acid status in relation to dietary intake.
Our data indicate that, by prescribing high-energy extra meals and/or energy supplements, total energy intake was higher than that recommended for age and in line to what is presently recommended for CF (3,4). The dietary questionnaire revealed that this was achieved by increasing lipid intake, particularly saturated and monounsaturated fatty acids, whereas the relative intake of polyunsaturated fats (including both the parental compounds—EFAs and their longer-chain derivatives, LCPUFA) was found to be lower than in control subjects.
Several studies have documented that the status of both EFA and LCPUFA is frequently poor in CF patients; however, very few data are available regarding the relationship between fatty acid status and dietary intakes, and this issue was investigated in our study. In line with the above-mentioned pattern of fat intake, also the plasma percentage levels of saturated and monounsaturated fatty acids also were higher in CF patients than controls, whereas those of PUFA (of both the n-3 and n-6 series) were lower. Overall, our data suggest that it is quite difficult to increase the intake of PUFA within the current dietary recommendations, and this is also the case when the dietary supplements presently available on the market are consumed. Indeed, energy integrators available for our patients do not allow for any significant increase in the intake of the polyunsaturated fatty acid fraction, and most of them include only carbohydrates and proteins. It cannot be excluded that this factor may have contributed in determining the poor PUFA and n-3L CPUFA status already described as an important characteristic of the disease (38). Indeed, in agreement with previous observations, plasma LA and DHA were lower in our CF patients, whereas those of ARA were comparable to controls. In all patients, the poor LA status was associated with an increase in ETA, thus confirming the existence of a characteristic PUFA profile in CF, which can be only, in part, influenced by dietary fat intakes or fat malabsorption (39–41). Our patients were on pancreatic enzyme replacement therapy, with adequate correction of fat malabsorption, as indicated by control of clinical symptoms, normal steatocrit values (data not shown) and satisfactory growth rate. Although dietary PUFA intakes are relatively lower and consistent with the plasma fatty acid profile (lower LA and higher ETA percentage levels), our study confirms that the plasma PUFA profile described in CF does not correspond to the profile observed in typical EFA deficiency (a condition otherwise associated with a reduced eicosanoid production) because, as pointed out by others (39), plasma ARA levels are not reduced, but even mildly increased. Overall, this fatty acid pattern seems to be indicative of a defect in the regulation of EFA metabolism, possibly related to the CF basic defect (functional defect of the CFTR protein) (11,12,42). Indeed, CF patients with biochemical signs of EFA deficiency (high ETA levels and high ETA/ARA ratio) have been described despite normal growth and good nutritional status (10).
Alternatively, the abnormalities in EFA metabolism may be the consequence of a chronic status of inflammation and/or malnutrition. To further test this hypothesis, as a third step of the study, we looked for correlations between FA status and some functional outcomes of the disease. We could only find a few associations indicating that the Shwachnan score (a reliable index of a patient's overall condition according to general activity, physical examination, nutritional status and conventional chest x-ray findings) tended to be worse in the presence of increased ARA and total n-6 LCPUFA in circulating PL. We were not able to identify any association between circulating n-3 LCPUFA and outcomes. Preliminary data in CF patients have suggested possible beneficial effects of DHA and eicosapentaenoic acid supplementation (contrasting the metabolic effects of ARA and n-6 LCPUFA) (43) on clinical outcomes, including pulmonary function, inflammatory status, growth and Shwachman score (44–46). These data could be explained on the basis that improved conditions are associated with a decreased synthesis of proinflammatory eicosanoids and then a decreased utilization of ARA.
In conclusion, our data suggest that the quality of fat intake, the clinical phenotype and CF-associated biomechanisms are bound in a vicious circle and may all concur to create the “spectrum” of CF. The current practice on dietary intervention is effective in increasing energy supply in CF patients, but, unless a specific PUFA and/or n-3 LCPUFA (DHA particularly) supplementation is intentionally planned, this extra-energy intake is mainly provided by saturated and monounsaturated fats and/or carbohydrate supplements. The poor PUFA intake is associated with lower LA, higher ETA and normal ARA in plasma PL, which are suggestive of an adjunctive specific EFA defect in CF, possibly connected with the basic membrane defect. Finally, the clinical correlates might be also affected by the FA status, depending, in turn, on both the dietary- and disease-related background. Further research on the effects of specific PUFA and n-3 LCPUFA supplementations are needed to check the reversibility of this vicious circle. In the meantime, when planning a dietary enrichment of CF patients, “ad-hoc” PUFA supplementations (including EFA and LCPUFA) should be considered to balance the fat supply.
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