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Influence of lung transplantation on the essential fatty acid profile in cystic fibrosis
Prostaglandins, Leukotrienes and Essential Fatty Acids xxx (xxxx) xxxx
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Influence of lung transplantation on the essential fatty acid profile in cystic fibrosis Laurence Hanssensa, J. Duchateaub, S.A. Namanec, A. Malfrootd, C. Knoope, G. Casimira a
Hôpital Universitaire des Enfants Reine Fabiola, Avenue J.J. Crocq, 15,1020 Brussels, Belgium Hôpital Universitaire des Enfants Reine Fabiola - Institut de mucoviscidose de l'ULB -Université Libre de Bruxelles, Brussels, Belgium c Universitair Ziekenhuis Brussel (UZ Brussel) - Vrije Universiteit Brussel (VUB), Brussels, Belgium d Hôpital Universitaire Erasme - Institut de mucoviscidose de l'ULB - Université Libre de Bruxelles, Brussels, Belgium e Laboratoire de pédiatrie de l'Hôpital Universitaire des Enfants Reine Fabiola - Université Libre de Bruxelles (ULB), Brussels, Belgium b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cystic fibrosis Essential fatty acid Polyunsaturated fatty acids Lung transplantation
Lung transplantation is assumed to normalize essential fatty acid (EFA) profile in the plasma, described as abnormal in patients with cystic fibrosis (CF). This study sought to evaluate the EFA profile in both the plasma and erythrocyte membrane according to lung status by comparing CF patients with or without a lung transplant. A total of 50 homozygous F508del patients (33 CF patients [CF group] and 17 CF patients with a lung transplant [TX CF group]) were included. In comparison with the CF group, in the plasma, the levels of total n-3, α-linolenic, eicosapentaenoic, and docosahexaenoic acids were higher and the n-6/n-3 ratio was lower in the TX CF group. Yet, these differences were not observed in the erythrocyte membrane. This study supports that lung transplantation improves the EFA profile in the plasma but not in the erythrocyte membrane by means of the different mechanisms suggested in this article.
1. Introduction Cystic fibrosis (CF) is a recessive genetic disease resulting from mutations in the CF transmembrane conductance regulator (CFTR) gene. Despite marked improvements in CF treatment, pulmonary disease remains the main cause of morbidity and mortality. An abnormal essential fatty acid (EFA) profile has been documented in the blood and tissues of CF patients, including decreased concentrations of linoleic acid (LA, 18:2 n-6) and docosahexaenoic acid (DHA, C22:6 n-3), associated with increased concentrations of arachidonic acid (AA, C20:4 n-6) [1–6]. Their origin remains elusive. EFA deficiency has been presumed to be secondary to fat malabsorption or low dietary intake [7,8. As this deficiency has also been reported in well-nourished CF patients [9], other mechanisms have been described, such as the excessive oxidation of EFA as an energy source, increased metabolism of LA to AA associated with increased expression and activation of Δ5- and Δ6-desaturase enzymes, increased production of eicosanoids linked to inflammatory responses, higher lipid turnover rates in cell membranes, defective lipid incorporation into the plasma membrane, as well as ceramide deficiency [1,10–14]. Moreover, EFA deficiency is more marked in severe CF genotypes, reinforcing the association between EFA metabolism disturbances and the basic CF defect via the AMPK signaling pathway [14–16].
Pulmonary inflammation is responsible for the progressive lung destruction observed in CF. Omega-3 supplementation down-regulates the production of inflammatory mediators [17–19] and, consequently, provides certain clinical benefits [19–24]. Both eicosapentaenoic acid (EPA, C20:5 n-3) and DHA can competitively inhibit proinflammatory mediator formation derived from AA, thereby reducing immune cell activities. Moreover, EPA and DHA were shown to generate anti-inflammatory and inflammation-resolving mediators called resolvins, protectins, and maresins [25]. To our knowledge, only one work has so far studied the influence of lung transplantation [26] on the plasma EFA profile in CF patients. Therefore, we sought to evaluate the EFA profile both in plasma and erythrocyte membrane according to lung status by comparing CF patients with and without a lung transplant. 2. Patients and methods 2.1. Study population During a 1-month period, we included in the study 33 CF patients (CF group) and 17 CF patients with a lung transplant (TX CF group) of any age, from two CF care units that implement similar standard CF care protocols: Institut de mucoviscidose de l'ULB (Université Libre de
E-mail address: [email protected] (L. Hanssens). https://doi.org/10.1016/j.plefa.2020.102060 Received 15 September 2019; Received in revised form 5 December 2019; Accepted 21 January 2020 0952-3278/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Laurence Hanssens, et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, https://doi.org/10.1016/j.plefa.2020.102060
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Bruxelles) and Universitair Ziekenhuis Brussel (UZ Brussel), Belgium. All patients included in the study were clinically stable and homozygous for the F508del mutation.
Table 1 Population characteristics. Groups
CF group (n = 33)
TX CF group (n = 17)
P value
Age, years Gender ratio (M:F) FEV1%
18.0 (10.5) 21:12 69.8 (26.3) (n = 28) 80.1 (20.1) (n = 28) 46.0 (32.9) (n = 28) −0.8 (1.1)
32.5 (11.2) 9:8 84.2 (23.0)
0.002 0.465 0.069
86.7 (19.7)
0.283
74.1 (34.6)
0.013
−1.1 (1.3)
0.443
2233.9 (925.7) 834.2 (452.3) 1071.2 (441.8)
1592.3 (635.6) 466.8 (207.3) 852.8 (424.9)
0.024 0.002 0.186
345.9 (155.5) 248 (258) 1.2 (1.2) 131 (242) 213 (397) 9.6 (6.3) 3.2 (2.4) 61.3 (47.1)
244.6 (93.4) 221 (334) 0.7 (0.6) 47 (71) 79 (122) 6.9 (5.0) 0.7 (0.9) (n = 14) 9.4 (13.7) (n = 14) 7.6 (3.9) 7.7 (3.6) 0.6 (0.9) 8.5 (2.6)
0.022 0.752 0.176 0.291 0.285 0.241 <0.001 <0.001
2.2. Study design This was a prospective observational study. After the signing of a patient and/or parental informed consent form, we conducted blood analyses of the plasma and erythrocyte membrane EFA profiles (total n3, α-linolenic acid [ALA, C18:3 n-3], EPA, DHA, total n-6, LA, AA, n-6/ n-3 ratio, and AA/DHA ratio) as well as oleic [C18:1 n-9] and palmitic [C16:0] acids, expressed as percentages of total fatty acids. The EFA profiles were quantified using gas chromatography (HP6890; HewlettPackard-Agilent) and mass spectrometry (MS 5973; Agilent Technologies) [8]. Inflammatory marker assessments (white blood cells [WBCs], polymorphonuclear neutrophils [PMNs], C-reactive protein [CRP], and immunoglobulin G [IgG]) were performed at the same time as the EFA measurements. Clinical parameters were evaluated for each patient; lung function was assessed only for patients older than 6 years. Clinical parameters comprised the patients’ age and gender, body mass index z-score (BMI z-score), adjusted for age and gender according to the Centers for Disease Control and Prevention (CDC) guidelines [27], cumulative number of pulmonary exacerbations (PEs), and duration of antibiotic therapy, measured in days over the last 12 months. A PE was defined as an acute episode requiring antibiotic therapy by any administration route during the study period. Lung function tests were performed according to the American Thoracic Society (ATS) and European Respiratory Society (ERS) guidelines [28]. Forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and forced expiratory flow 25–75% (FEF25-75) were expressed in percentages of predicted values [29]. The daily average of total energy, lipid, carbohydrate and protein intakes, as well as that of ALA, EPA, DHA, and LA intakes, and the weekly average of fatty fish intake were calculated based on a 3-day diary and food frequency questionnaire (FFQ).
FVC% FEF25-75% BMI z-score Dietary intake Energy intake, kcal/day Lipid intake, kcal/day Carbohydrate intake, kcal/ day Protein intake, kcal/day Fatty fish intake, g/week ALA intake, g/day EPA intake, mg/day DHA intake, mg/day LA intake, g/day N exacerbations Days of ATB WBC, 103 /mm3 PMNs, 103 /mm3 CRP, mg/dl IgG, g/L
9.4 (3.4) 5.8 (2.9) 0.8 (0.6) 13.3 (5.4)
0.103 0.988 0.472 <0.001
Abbreviations: Gender ratio (M:F): Male:Female; FEV1%: forced expiratory volume in percentage of predicted value; FVC%: forced vital capacity as percentage predicted value; FEF25-75%: forced expiratory flow 25–75% as percentage predicted value; BMI: body mass index; N exacerbations: number of exacerbations; ATB: antibiotics; WBCs: white blood cells; PMNs: polymorphonuclear neutrophils; CRP: C-reactive protein; IgG: immunoglobulin G.Data have been expressed as mean ± standard deviation; a P value of ≤0.05 was considered statistically significant.
2.3. Statistical analysis FEF25-75% was higher in the TX CF group, while the total energy, lipid and protein intakes were higher in the CF group. As inflammation markers, WBC, PMN, and CRP levels were not different. The mean IgG levels were notably increased above normal values in the CF group, whereas they were significantly reduced compared with the expected normal range in the TX CF group. In order to determine the cumulative number of PEs and duration of antibiotic therapy within the last 12 months, only patients who underwent a lung transplantation performed at least 1 year ago were included in the analysis (n = 14). The cumulative number of PEs and duration of antibiotic therapy, measured in days during the last 12 months, were both lower in the TX CF group.
The statistical analyses were carried out using the Analyse-it® 3.90.7 software for Microsoft Excel (Analyse-it Software, Ltd., Leeds, United Kingdom), according to the manufacturer's instructions. Continuous data were expressed as mean ± standard deviation or as median and interquartile range, as appropriate. Categorical variables were expressed as percentages. We performed the Student's t-test as the parametric assessment and Mann–Whitney U test as the non-parametric assessment for intergroup comparisons. To compare the categorical variables, the chi-squared test for two-way tables (or the Fisher's exact test) was applied. The Spearman rank correlation coefficient was employed for correlations. A p value ≤0.05 was considered statistically significant.
3.2. EFA profiles and lung status
2.4. Ethical committee
The plasma and erythrocyte membrane EFA levels according to lung status (CF group vs. TX CF group) have been presented in Tables 2 and 3. In the plasma, the EFA profile significantly differed between the two groups. Compared to the CF group, the total n-3, EPA, and DHA levels were higher, and n-6/n-3 ratio was lower in the TX CF group. In the erythrocyte membrane, these differences were not observed. No correlation was shown between the duration of lung transplantation and EFA profile.
This study was approved by the university hospital's ethical committee (HUDERF). 3. Results 3.1. Population characteristics A total of 50 patients were included in the study: 33 CF patients without a lung transplant (CF group) and 17 with a lung transplant (TX CF group) (Table 1). All patients were suffering from exocrine pancreatic insufficiency. The TX CF group was older in age. The duration of lung transplantation was 5.3 years (0.4–14.2). No significant differences were observed between the two groups in terms of gender, BMI zscore, carbohydrate intake, weekly fish intake, FEV1%, and FVC%, but
3.3. EFA profiles and clinical and inflammatory variables In both the CF and TX CF groups, no correlation was observed between the EFA profiles in the plasma and erythrocyte membrane and patients’ age, cumulative number of PEs, duration of antibiotic therapy, 2
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their analysis only on plasma [26]. Therefore, we sought to evaluate the EFA profile in plasma as well as in the erythrocyte membrane according to lung status by comparing CF patients with and without a lung transplant. As EFA deficiency has been reported to particularly occur in CF patients with severe mutations [14–16], our study included only those who were homozygous for the F508del mutation. According to lung status, the EFA profile significantly differed, but only in the plasma. Indeed, compared to the CF patients without a lung transplant, the plasma EFA profile was better in CF patients with a lung transplant, as they presented higher levels of total n-3, EPA, and DHA and lower levels of the n-6/n-3 ratio. Witters et al. described a normalization of LA, DHA, and AA concentrations in the plasma of CF patients with a lung transplant; the authors hypothesized that the CF lung is a major contributor towards an abnormal fatty acid profile [26]. Given that we observed in our study that the inflammatory status, as expressed by IgG levels, significantly improved in CF patients with a lung transplant and that the duration of the lung transplant, patients’ age, and EFA food intake did not account for these differences in the plasma, we clearly support the mechanisms suggested by Witters et al. and others. The inflammatory profiles due to the removal of infected and inflamed CF lungs, excessive EFA oxidation as an energy source, and increased LA to AA metabolism, which all are highly attenuated or even no longer present following lung transplantation, could explain this improvement in the plasma EFA profile, in addition to the presence of functional CFTR in the lungs of CF patients with a lung transplant, along with the effects of immunosuppressive therapy [1,10–16,26]. To the best of our knowledge, only our study and that of Witters et al. have compared the EFA profiles in CF patients with and without a lung transplant. However, in our study, we also assessed the EFA profiles in the erythrocyte membrane. The plasma fatty acid levels have been described as markers to detect EFA status deterioration at an early time point, while correlating well to those measured in the erythrocyte membrane [2]. However, the determination of the fatty acid profile in the erythrocyte membrane is considered the most reliable marker, reflecting the intake over several months [2]. In this study, while the EFA profile in CF patients with a lung transplant was shown to be superior in the plasma, these differences were not observed in the erythrocyte membrane. The duration of lung transplantation and patients’ age did not explain these paradoxical differences in EFA profiles between the plasma and erythrocyte membrane. While the total energy and lipid intakes were higher in the CF group, the EFA profile was superior only in the plasma of the TX CF group. The lack of differences in the EFA profiles in the erythrocyte membrane according to lung status could be explained by the studies involving omega-3 supplementation. Indeed, the high doses of EFA employed in these studies were associated with increased EFA levels, both in the plasma and erythrocyte membrane of CF patients without a lung transplant, along with a decrease in inflammatory mediators [17–19], in addition to clinical benefits [19–24]. As the energy and EFA consumptions are excessive in CF patients, only omega-3 supplementation would be likely to improve the gradients between the intestinal lumen, plasma, and cell membrane and, therefore, the EFA levels in the plasma as well as in the erythrocyte membrane of both CF patients with and without a transplant. By removing infected and inflamed CF lungs, this improvement would probably be more marked in CF patients with a transplant. The major limitation of our study is that the CF patients with a lung transplant were not matched for age; thus, the ages in both groups were significantly different. Another limitation is that we did not take samples both before and after lung transplantation. The last limitation is that the population in our study was small in size. In conclusion, as demonstrated in this study, the EFA profile was shown to improve in the plasma improves following lung transplantation; therefore, CF lung is likely to be an important contributor towards abnormal EFA profiles in CF patients. Even if the exact mechanism remains elusive, higher doses of omega-3 supplementation could probably improve the EFA profile in the erythrocyte membrane.
Table 2 Plasma levels of EFA according to lung status (CF patients vs. CF lung transplant patients) . EFA (% total FA)
CF group (n = 33) median (IQR)
TX CF group (n = 17) median (IQR)
P value
Total n-3a C18:3 n-3 (ALA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) Total n-6b C18:2 n-6 (LA) C20:4 n-6 (AA) AA/DHA ratio n-6b/n-3a ratio
3.11 0.47 0.69 1.23
3.99 0.62 0.91 1.88
0.004 0.180 0.020 0.011
(2.55–3.85) (0.31–0.65) (0.46–0.96) (1.02–1.84)
32.17 (28.83–34.54) 22.42 (18.87–24.58) 6.99 (6.04–7.94) 5.06 (4.29–6.37) 9.91 (8.29–12.89)
(3.32–4.91) (0.42–0.71) (0.74–1.51) (1.44–2.42)
33.66 (28.13–34.51) 21.25 (19.04–23.56) 7.74 (6.52–9.10) 4.36 (3.30–5.97) 7.94 (6.47–9.56)
0.943 0.652 0.110 0.117 0.004
Abbreviations: EFA: essential fatty acids; FA: fatty acids; IQR: interquartile range; CF group: cystic fibrosis patients; TX CF group: CF patients with a lung transplant; NS: not significant. Data have been expressed as percentages of total fatty acids; a P value of ≤0.05 was considered statistically significant. Compared to the CF group, the total n-3, EPA, and DHA levels were higher, and n-6/n-3 ratio was lower in the TX CF group. No difference was observed between the two groups for oleic and palmitic acids. a Total amount of pooled levels of n-3 PUFA: C18:3 n-3 (ALA) + C20:5 n-3 (EPA) + C22:5 n-3 (DPA) + C22:6 n-3 (DHA). b Total amount of pooled levels of n-6 PUFA: C18:2 n-6 (LA) + C18:3 n-6 (GLA) + C20:3 n-6 (DGLA) + C20:4 n-6 (AA) + C22:4 n-6 (DTA) + C22:5 n-6 (DPA). Table 3 EFA erythrocyte membrane levels according to lung status (CF patients vs. CF lung transplant patients). EFA (% total FA)
CF group (n = 33) median (IQR)
TX CF group (n = 17) median (IQR)
P value
Total n-3a C18:3 n-3 (ALA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) Total n-6b C18:2 n-6 (LA) C20:4 n-6 (AA) AA/DHA ratio n-6b/n-3a ratio
6.62 (4.44–7.87) 0 (0–0.04) 0.64 (0.36–0.79) 3.54 (1.63–4.11)
7.47 0.01 0.66 3.48
0.227 0.782 0.073 0.341
28.62 (22.70–30.45) 7.30 (5.90–8.43) 16.32 (12.34–17.29) 4.88 (3.73–6.57) 4.40 (3.75–6.83)
28.86 (27.44–30.12) 7.02 (6.56–7.84) 17.28 (15.14–18.24) 5.24 (3.57–5.70) 4.35 (3.55–4.69)
(6.23–8.34) (0–0.03) (0.57–0.88) (3.12–4.49)
0.814 0.927 0.062 0.878 0.282
Abbreviations: EFA: essential fatty acids; FA: fatty acids; IQR: interquartile range; CF group: cystic fibrosis patients; TX CF group: CF patients with a lung transplant; NS: not significant. Data have been expressed as percentages of total fatty acids; a P value of ≤0.05 was considered statistically significant. No difference was observed between the two groups for EFA as well as for oleic and palmitic acids. a Total amount of pooled levels of n-3 PUFA: C18:3 n-3 (ALA) + C20:5 n-3 (EPA) + C22:5 n-3 (DPA) + C22:6 n-3 (DHA). b Total amount of pooled levels of n-6 PUFA: C18:2 n-6 (LA) + C18:3 n-6 (GLA) + C20:3 n-6 (DGLA) + C20:4 n-6 (AA) + C22:4 n-6 (DTA) + C22:5 n-6 (DPA).
or inflammatory parameters. 4. Discussion and conclusions More than 50 years ago, Kuo et al. first described the abnormal fatty acid profile in CF [30]. This abnormality has since been confirmed, particularly for EFA [1–5,14]. The most common alterations are decreased concentrations of LA, whereas decreased concentrations of DHA and increased concentrations of AA have also been described. The origin of this abnormal EFA profile remains elusive. To our knowledge, only one work has so far studied the influence of lung transplantation [26] on the EFA profile in CF patients. Witters et al. observed that lung transplantation normalized patients’ EFA profile, but they performed 3
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CRediT authorship contribution statement [11]
Laurence Hanssens: Conceptualization, Data curation, Formal analysis, Funding acquisition. J. Duchateau: . S.A. Namane: . A. Malfroot: . C. Knoop: . G. Casimir: .
[12]
Declaration of Competing Interest
[13]
The authors state that they have no conflicts of interest to declare.
[14]
Acknowledgements [15]
This study was supported by a grant from the Belgian Cystic Fibrosis Association. We are grateful to Térésinha Leal and her team for the EFA analysis as well as to Isabelle Thiébaut, Veronique Gaspar, and Bernard Wenderickx for their generous contributions. We gratefully acknowledge the participation in this study of all the CF patients and their families.
[16]
[17]
[18]
Supplementary materials [19]
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2020.102060.
[20]
[21]
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Influence of lung transplantation on the essential fatty acid profile in cystic fibrosis Laurence Hanssensa, J. Duchateaub, S.A. Namanec, A. Malfrootd, C. Knoope, G. Casimira a
Hôpital Universitaire des Enfants Reine Fabiola, Avenue J.J. Crocq, 15,1020 Brussels, Belgium Hôpital Universitaire des Enfants Reine Fabiola - Institut de mucoviscidose de l'ULB -Université Libre de Bruxelles, Brussels, Belgium c Universitair Ziekenhuis Brussel (UZ Brussel) - Vrije Universiteit Brussel (VUB), Brussels, Belgium d Hôpital Universitaire Erasme - Institut de mucoviscidose de l'ULB - Université Libre de Bruxelles, Brussels, Belgium e Laboratoire de pédiatrie de l'Hôpital Universitaire des Enfants Reine Fabiola - Université Libre de Bruxelles (ULB), Brussels, Belgium b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cystic fibrosis Essential fatty acid Polyunsaturated fatty acids Lung transplantation
Lung transplantation is assumed to normalize essential fatty acid (EFA) profile in the plasma, described as abnormal in patients with cystic fibrosis (CF). This study sought to evaluate the EFA profile in both the plasma and erythrocyte membrane according to lung status by comparing CF patients with or without a lung transplant. A total of 50 homozygous F508del patients (33 CF patients [CF group] and 17 CF patients with a lung transplant [TX CF group]) were included. In comparison with the CF group, in the plasma, the levels of total n-3, α-linolenic, eicosapentaenoic, and docosahexaenoic acids were higher and the n-6/n-3 ratio was lower in the TX CF group. Yet, these differences were not observed in the erythrocyte membrane. This study supports that lung transplantation improves the EFA profile in the plasma but not in the erythrocyte membrane by means of the different mechanisms suggested in this article.
1. Introduction Cystic fibrosis (CF) is a recessive genetic disease resulting from mutations in the CF transmembrane conductance regulator (CFTR) gene. Despite marked improvements in CF treatment, pulmonary disease remains the main cause of morbidity and mortality. An abnormal essential fatty acid (EFA) profile has been documented in the blood and tissues of CF patients, including decreased concentrations of linoleic acid (LA, 18:2 n-6) and docosahexaenoic acid (DHA, C22:6 n-3), associated with increased concentrations of arachidonic acid (AA, C20:4 n-6) [1–6]. Their origin remains elusive. EFA deficiency has been presumed to be secondary to fat malabsorption or low dietary intake [7,8. As this deficiency has also been reported in well-nourished CF patients [9], other mechanisms have been described, such as the excessive oxidation of EFA as an energy source, increased metabolism of LA to AA associated with increased expression and activation of Δ5- and Δ6-desaturase enzymes, increased production of eicosanoids linked to inflammatory responses, higher lipid turnover rates in cell membranes, defective lipid incorporation into the plasma membrane, as well as ceramide deficiency [1,10–14]. Moreover, EFA deficiency is more marked in severe CF genotypes, reinforcing the association between EFA metabolism disturbances and the basic CF defect via the AMPK signaling pathway [14–16].
Pulmonary inflammation is responsible for the progressive lung destruction observed in CF. Omega-3 supplementation down-regulates the production of inflammatory mediators [17–19] and, consequently, provides certain clinical benefits [19–24]. Both eicosapentaenoic acid (EPA, C20:5 n-3) and DHA can competitively inhibit proinflammatory mediator formation derived from AA, thereby reducing immune cell activities. Moreover, EPA and DHA were shown to generate anti-inflammatory and inflammation-resolving mediators called resolvins, protectins, and maresins [25]. To our knowledge, only one work has so far studied the influence of lung transplantation [26] on the plasma EFA profile in CF patients. Therefore, we sought to evaluate the EFA profile both in plasma and erythrocyte membrane according to lung status by comparing CF patients with and without a lung transplant. 2. Patients and methods 2.1. Study population During a 1-month period, we included in the study 33 CF patients (CF group) and 17 CF patients with a lung transplant (TX CF group) of any age, from two CF care units that implement similar standard CF care protocols: Institut de mucoviscidose de l'ULB (Université Libre de
E-mail address: [email protected] (L. Hanssens). https://doi.org/10.1016/j.plefa.2020.102060 Received 15 September 2019; Received in revised form 5 December 2019; Accepted 21 January 2020 0952-3278/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Laurence Hanssens, et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, https://doi.org/10.1016/j.plefa.2020.102060
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Bruxelles) and Universitair Ziekenhuis Brussel (UZ Brussel), Belgium. All patients included in the study were clinically stable and homozygous for the F508del mutation.
Table 1 Population characteristics. Groups
CF group (n = 33)
TX CF group (n = 17)
P value
Age, years Gender ratio (M:F) FEV1%
18.0 (10.5) 21:12 69.8 (26.3) (n = 28) 80.1 (20.1) (n = 28) 46.0 (32.9) (n = 28) −0.8 (1.1)
32.5 (11.2) 9:8 84.2 (23.0)
0.002 0.465 0.069
86.7 (19.7)
0.283
74.1 (34.6)
0.013
−1.1 (1.3)
0.443
2233.9 (925.7) 834.2 (452.3) 1071.2 (441.8)
1592.3 (635.6) 466.8 (207.3) 852.8 (424.9)
0.024 0.002 0.186
345.9 (155.5) 248 (258) 1.2 (1.2) 131 (242) 213 (397) 9.6 (6.3) 3.2 (2.4) 61.3 (47.1)
244.6 (93.4) 221 (334) 0.7 (0.6) 47 (71) 79 (122) 6.9 (5.0) 0.7 (0.9) (n = 14) 9.4 (13.7) (n = 14) 7.6 (3.9) 7.7 (3.6) 0.6 (0.9) 8.5 (2.6)
0.022 0.752 0.176 0.291 0.285 0.241 <0.001 <0.001
2.2. Study design This was a prospective observational study. After the signing of a patient and/or parental informed consent form, we conducted blood analyses of the plasma and erythrocyte membrane EFA profiles (total n3, α-linolenic acid [ALA, C18:3 n-3], EPA, DHA, total n-6, LA, AA, n-6/ n-3 ratio, and AA/DHA ratio) as well as oleic [C18:1 n-9] and palmitic [C16:0] acids, expressed as percentages of total fatty acids. The EFA profiles were quantified using gas chromatography (HP6890; HewlettPackard-Agilent) and mass spectrometry (MS 5973; Agilent Technologies) [8]. Inflammatory marker assessments (white blood cells [WBCs], polymorphonuclear neutrophils [PMNs], C-reactive protein [CRP], and immunoglobulin G [IgG]) were performed at the same time as the EFA measurements. Clinical parameters were evaluated for each patient; lung function was assessed only for patients older than 6 years. Clinical parameters comprised the patients’ age and gender, body mass index z-score (BMI z-score), adjusted for age and gender according to the Centers for Disease Control and Prevention (CDC) guidelines [27], cumulative number of pulmonary exacerbations (PEs), and duration of antibiotic therapy, measured in days over the last 12 months. A PE was defined as an acute episode requiring antibiotic therapy by any administration route during the study period. Lung function tests were performed according to the American Thoracic Society (ATS) and European Respiratory Society (ERS) guidelines [28]. Forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and forced expiratory flow 25–75% (FEF25-75) were expressed in percentages of predicted values [29]. The daily average of total energy, lipid, carbohydrate and protein intakes, as well as that of ALA, EPA, DHA, and LA intakes, and the weekly average of fatty fish intake were calculated based on a 3-day diary and food frequency questionnaire (FFQ).
FVC% FEF25-75% BMI z-score Dietary intake Energy intake, kcal/day Lipid intake, kcal/day Carbohydrate intake, kcal/ day Protein intake, kcal/day Fatty fish intake, g/week ALA intake, g/day EPA intake, mg/day DHA intake, mg/day LA intake, g/day N exacerbations Days of ATB WBC, 103 /mm3 PMNs, 103 /mm3 CRP, mg/dl IgG, g/L
9.4 (3.4) 5.8 (2.9) 0.8 (0.6) 13.3 (5.4)
0.103 0.988 0.472 <0.001
Abbreviations: Gender ratio (M:F): Male:Female; FEV1%: forced expiratory volume in percentage of predicted value; FVC%: forced vital capacity as percentage predicted value; FEF25-75%: forced expiratory flow 25–75% as percentage predicted value; BMI: body mass index; N exacerbations: number of exacerbations; ATB: antibiotics; WBCs: white blood cells; PMNs: polymorphonuclear neutrophils; CRP: C-reactive protein; IgG: immunoglobulin G.Data have been expressed as mean ± standard deviation; a P value of ≤0.05 was considered statistically significant.
2.3. Statistical analysis FEF25-75% was higher in the TX CF group, while the total energy, lipid and protein intakes were higher in the CF group. As inflammation markers, WBC, PMN, and CRP levels were not different. The mean IgG levels were notably increased above normal values in the CF group, whereas they were significantly reduced compared with the expected normal range in the TX CF group. In order to determine the cumulative number of PEs and duration of antibiotic therapy within the last 12 months, only patients who underwent a lung transplantation performed at least 1 year ago were included in the analysis (n = 14). The cumulative number of PEs and duration of antibiotic therapy, measured in days during the last 12 months, were both lower in the TX CF group.
The statistical analyses were carried out using the Analyse-it® 3.90.7 software for Microsoft Excel (Analyse-it Software, Ltd., Leeds, United Kingdom), according to the manufacturer's instructions. Continuous data were expressed as mean ± standard deviation or as median and interquartile range, as appropriate. Categorical variables were expressed as percentages. We performed the Student's t-test as the parametric assessment and Mann–Whitney U test as the non-parametric assessment for intergroup comparisons. To compare the categorical variables, the chi-squared test for two-way tables (or the Fisher's exact test) was applied. The Spearman rank correlation coefficient was employed for correlations. A p value ≤0.05 was considered statistically significant.
3.2. EFA profiles and lung status
2.4. Ethical committee
The plasma and erythrocyte membrane EFA levels according to lung status (CF group vs. TX CF group) have been presented in Tables 2 and 3. In the plasma, the EFA profile significantly differed between the two groups. Compared to the CF group, the total n-3, EPA, and DHA levels were higher, and n-6/n-3 ratio was lower in the TX CF group. In the erythrocyte membrane, these differences were not observed. No correlation was shown between the duration of lung transplantation and EFA profile.
This study was approved by the university hospital's ethical committee (HUDERF). 3. Results 3.1. Population characteristics A total of 50 patients were included in the study: 33 CF patients without a lung transplant (CF group) and 17 with a lung transplant (TX CF group) (Table 1). All patients were suffering from exocrine pancreatic insufficiency. The TX CF group was older in age. The duration of lung transplantation was 5.3 years (0.4–14.2). No significant differences were observed between the two groups in terms of gender, BMI zscore, carbohydrate intake, weekly fish intake, FEV1%, and FVC%, but
3.3. EFA profiles and clinical and inflammatory variables In both the CF and TX CF groups, no correlation was observed between the EFA profiles in the plasma and erythrocyte membrane and patients’ age, cumulative number of PEs, duration of antibiotic therapy, 2
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their analysis only on plasma [26]. Therefore, we sought to evaluate the EFA profile in plasma as well as in the erythrocyte membrane according to lung status by comparing CF patients with and without a lung transplant. As EFA deficiency has been reported to particularly occur in CF patients with severe mutations [14–16], our study included only those who were homozygous for the F508del mutation. According to lung status, the EFA profile significantly differed, but only in the plasma. Indeed, compared to the CF patients without a lung transplant, the plasma EFA profile was better in CF patients with a lung transplant, as they presented higher levels of total n-3, EPA, and DHA and lower levels of the n-6/n-3 ratio. Witters et al. described a normalization of LA, DHA, and AA concentrations in the plasma of CF patients with a lung transplant; the authors hypothesized that the CF lung is a major contributor towards an abnormal fatty acid profile [26]. Given that we observed in our study that the inflammatory status, as expressed by IgG levels, significantly improved in CF patients with a lung transplant and that the duration of the lung transplant, patients’ age, and EFA food intake did not account for these differences in the plasma, we clearly support the mechanisms suggested by Witters et al. and others. The inflammatory profiles due to the removal of infected and inflamed CF lungs, excessive EFA oxidation as an energy source, and increased LA to AA metabolism, which all are highly attenuated or even no longer present following lung transplantation, could explain this improvement in the plasma EFA profile, in addition to the presence of functional CFTR in the lungs of CF patients with a lung transplant, along with the effects of immunosuppressive therapy [1,10–16,26]. To the best of our knowledge, only our study and that of Witters et al. have compared the EFA profiles in CF patients with and without a lung transplant. However, in our study, we also assessed the EFA profiles in the erythrocyte membrane. The plasma fatty acid levels have been described as markers to detect EFA status deterioration at an early time point, while correlating well to those measured in the erythrocyte membrane [2]. However, the determination of the fatty acid profile in the erythrocyte membrane is considered the most reliable marker, reflecting the intake over several months [2]. In this study, while the EFA profile in CF patients with a lung transplant was shown to be superior in the plasma, these differences were not observed in the erythrocyte membrane. The duration of lung transplantation and patients’ age did not explain these paradoxical differences in EFA profiles between the plasma and erythrocyte membrane. While the total energy and lipid intakes were higher in the CF group, the EFA profile was superior only in the plasma of the TX CF group. The lack of differences in the EFA profiles in the erythrocyte membrane according to lung status could be explained by the studies involving omega-3 supplementation. Indeed, the high doses of EFA employed in these studies were associated with increased EFA levels, both in the plasma and erythrocyte membrane of CF patients without a lung transplant, along with a decrease in inflammatory mediators [17–19], in addition to clinical benefits [19–24]. As the energy and EFA consumptions are excessive in CF patients, only omega-3 supplementation would be likely to improve the gradients between the intestinal lumen, plasma, and cell membrane and, therefore, the EFA levels in the plasma as well as in the erythrocyte membrane of both CF patients with and without a transplant. By removing infected and inflamed CF lungs, this improvement would probably be more marked in CF patients with a transplant. The major limitation of our study is that the CF patients with a lung transplant were not matched for age; thus, the ages in both groups were significantly different. Another limitation is that we did not take samples both before and after lung transplantation. The last limitation is that the population in our study was small in size. In conclusion, as demonstrated in this study, the EFA profile was shown to improve in the plasma improves following lung transplantation; therefore, CF lung is likely to be an important contributor towards abnormal EFA profiles in CF patients. Even if the exact mechanism remains elusive, higher doses of omega-3 supplementation could probably improve the EFA profile in the erythrocyte membrane.
Table 2 Plasma levels of EFA according to lung status (CF patients vs. CF lung transplant patients) . EFA (% total FA)
CF group (n = 33) median (IQR)
TX CF group (n = 17) median (IQR)
P value
Total n-3a C18:3 n-3 (ALA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) Total n-6b C18:2 n-6 (LA) C20:4 n-6 (AA) AA/DHA ratio n-6b/n-3a ratio
3.11 0.47 0.69 1.23
3.99 0.62 0.91 1.88
0.004 0.180 0.020 0.011
(2.55–3.85) (0.31–0.65) (0.46–0.96) (1.02–1.84)
32.17 (28.83–34.54) 22.42 (18.87–24.58) 6.99 (6.04–7.94) 5.06 (4.29–6.37) 9.91 (8.29–12.89)
(3.32–4.91) (0.42–0.71) (0.74–1.51) (1.44–2.42)
33.66 (28.13–34.51) 21.25 (19.04–23.56) 7.74 (6.52–9.10) 4.36 (3.30–5.97) 7.94 (6.47–9.56)
0.943 0.652 0.110 0.117 0.004
Abbreviations: EFA: essential fatty acids; FA: fatty acids; IQR: interquartile range; CF group: cystic fibrosis patients; TX CF group: CF patients with a lung transplant; NS: not significant. Data have been expressed as percentages of total fatty acids; a P value of ≤0.05 was considered statistically significant. Compared to the CF group, the total n-3, EPA, and DHA levels were higher, and n-6/n-3 ratio was lower in the TX CF group. No difference was observed between the two groups for oleic and palmitic acids. a Total amount of pooled levels of n-3 PUFA: C18:3 n-3 (ALA) + C20:5 n-3 (EPA) + C22:5 n-3 (DPA) + C22:6 n-3 (DHA). b Total amount of pooled levels of n-6 PUFA: C18:2 n-6 (LA) + C18:3 n-6 (GLA) + C20:3 n-6 (DGLA) + C20:4 n-6 (AA) + C22:4 n-6 (DTA) + C22:5 n-6 (DPA). Table 3 EFA erythrocyte membrane levels according to lung status (CF patients vs. CF lung transplant patients). EFA (% total FA)
CF group (n = 33) median (IQR)
TX CF group (n = 17) median (IQR)
P value
Total n-3a C18:3 n-3 (ALA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) Total n-6b C18:2 n-6 (LA) C20:4 n-6 (AA) AA/DHA ratio n-6b/n-3a ratio
6.62 (4.44–7.87) 0 (0–0.04) 0.64 (0.36–0.79) 3.54 (1.63–4.11)
7.47 0.01 0.66 3.48
0.227 0.782 0.073 0.341
28.62 (22.70–30.45) 7.30 (5.90–8.43) 16.32 (12.34–17.29) 4.88 (3.73–6.57) 4.40 (3.75–6.83)
28.86 (27.44–30.12) 7.02 (6.56–7.84) 17.28 (15.14–18.24) 5.24 (3.57–5.70) 4.35 (3.55–4.69)
(6.23–8.34) (0–0.03) (0.57–0.88) (3.12–4.49)
0.814 0.927 0.062 0.878 0.282
Abbreviations: EFA: essential fatty acids; FA: fatty acids; IQR: interquartile range; CF group: cystic fibrosis patients; TX CF group: CF patients with a lung transplant; NS: not significant. Data have been expressed as percentages of total fatty acids; a P value of ≤0.05 was considered statistically significant. No difference was observed between the two groups for EFA as well as for oleic and palmitic acids. a Total amount of pooled levels of n-3 PUFA: C18:3 n-3 (ALA) + C20:5 n-3 (EPA) + C22:5 n-3 (DPA) + C22:6 n-3 (DHA). b Total amount of pooled levels of n-6 PUFA: C18:2 n-6 (LA) + C18:3 n-6 (GLA) + C20:3 n-6 (DGLA) + C20:4 n-6 (AA) + C22:4 n-6 (DTA) + C22:5 n-6 (DPA).
or inflammatory parameters. 4. Discussion and conclusions More than 50 years ago, Kuo et al. first described the abnormal fatty acid profile in CF [30]. This abnormality has since been confirmed, particularly for EFA [1–5,14]. The most common alterations are decreased concentrations of LA, whereas decreased concentrations of DHA and increased concentrations of AA have also been described. The origin of this abnormal EFA profile remains elusive. To our knowledge, only one work has so far studied the influence of lung transplantation [26] on the EFA profile in CF patients. Witters et al. observed that lung transplantation normalized patients’ EFA profile, but they performed 3
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CRediT authorship contribution statement [11]
Laurence Hanssens: Conceptualization, Data curation, Formal analysis, Funding acquisition. J. Duchateau: . S.A. Namane: . A. Malfroot: . C. Knoop: . G. Casimir: .
[12]
Declaration of Competing Interest
[13]
The authors state that they have no conflicts of interest to declare.
[14]
Acknowledgements [15]
This study was supported by a grant from the Belgian Cystic Fibrosis Association. We are grateful to Térésinha Leal and her team for the EFA analysis as well as to Isabelle Thiébaut, Veronique Gaspar, and Bernard Wenderickx for their generous contributions. We gratefully acknowledge the participation in this study of all the CF patients and their families.
[16]
[17]
[18]
Supplementary materials [19]
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2020.102060.
[20]
[21]
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