Imp. 1/03

Ped. Med. Chir. (Med. Surg. Ped.), 2003, 25: 42-49 Fatty acids composition of plasma phospholipids
and triglycerides in children with cystic fibrosis.
The effect of dietary supplementation with
an olive and soybean oils mixture
Composizione in acidi grassi dei fosfolipidi plasmatici e dei trigliceridi in bambini con fibrosi cistica. Effetto di un’integrazione alimentare con una miscela di olio di oliva e soia Caramia G.1, Cocchi M.2, Gagliardini R.3, Malavolta M.4, Mozzon M.4, Frega N.G.4
Riassunto
nuzione significativa degli acidi grassi saturi (C 16:0, C 17:0,C 18:0, C 22:0). Inoltre, il rapporto tra LA e AA è aumentato La maggior parte dei pazienti affetti da fibrosi cistica presen- significativamente nel gruppo che faceva uso del supplemen- ta una carenza di acidi grassi essenziali (EFA) particolarmente to dietetico.Anche nei fosfolipidi del gruppo supplementato evidente nei fosfolipidi plasmatici. È noto da tempo che la con la miscela d’olio la percentuale relativa di C 18:1 è ridotta disponibilità di EFA modifica profondamente la distri- aumentata significativamente, così come quella di acido pal- buzione degli acidi grassi nelle diverse classi lipidiche pla- mitico, mentre sono diminuite le percentuali relative dei più smatiche e nei tessuti e può determinare profondi cambia- importati acidi grassi polinsaturi (PUFA). Questi risultati evi- menti nella fluidità di membrana e nei meccanismi di comu- denziano come l’acido oleico possa essere facilmente assor- nicazione cellulare. Nel presente studio sono presentati i bito e incorporato nei fosfolipidi plasmatici dei pazienti con risultati di una nuova strategia mirata alla realizzazione di un CF, con insufficienza pancreatica ma che fanno uso di enzimi integratore di acidi grassi facilmente reperibile e a basso pancreatici, mentre l’incorporazione di LA risulta meno evi- costo, che possa essere impiegato quotidianamente dai dente.A livello clinico è stato notato che, nonostante sia evi- pazienti affetti da CF con carenze di EFA.A tal fine, è stata stu- dente una riduzione di PUFA nei fosfolipidi plasmatici, i sog- diata la composizione in acidi grassi dei fosfolipidi e dei tri- getti sottoposti alla dieta sperimentale non hanno riportato gliceridi plasmatici, in pazienti che facevano uso di un sup- modificazioni significative nel quadro patologico complessi- plemento dietetico costituito da una miscela di olio di soja vo. Non sono state evidenziate differenze tra i due gruppi in (50%) e di olio extravergine di oliva (50%), e ne sono stati nessuno degli indici clinici monitorati (altezza, peso, BMI, test valutati gli aspetti clinici. Lo studio comprendeva 14 soggetti, affetti da CF, di età compresa tra i 6 e i 15 anni con insuffi-cienza pancreatica e portatori di uno o due alleli per la muta-zione ∆F508. I soggetti sono stati accoppiati in base all’età e Abstract
assegnati casualmente ad un gruppo supplementato con lamiscela d’olio (n = 7) o a un gruppo di controllo (n = 7).A dif- Cystic fibrosis (CF) is characterized by abnormal levels
ferenza del gruppo di controllo i pazienti che facevano uso of essential fatty acids (EFA) in plasma phospholipids.
della miscela d’olio hanno mostrato un aumento significativo The reduced availability of EFA has been reported to
della percentuale relativa di C 18:1 nei trigliceridi e una dimi- alter patterns of circulating and tissue esterified acids
and may determine profound changes in membrane
fluidity and cell signaling mechanisms. In the current
1Primario Emerito di Pediatria e Neonatologia - Azienda Ospedaliera “G. Salesi” - study, the results of a new strategy aimed at the real-
ization of a practical, low cost integrator, for daily use
Department of Biochemical Sciences - Scottish Agricultural College - Auchincruive in the dietary management of FC subjects, are report-
3Azienda Ospedaliera “G. Salesi” - Ancona ed. We investigated the plasma phospholipids and
4Dipartimento di Biotecnologie Agrarie ed Ambientali - Facoltà di Agraria - Università triglycerides fatty acids composition of CF patients
subjected to a dietary supplement constituted of a mix-
Indirizzo per la corrispondenza (Corresponding author): Università degli Studi di ture of 50% extra virgin olive oil and 50% soybean oil
Ancona - Facoltà di Agraria - Via Brecce Bianche - 60131 Ancona - tel. 071/2204924 -fax 071/2204980 - e-mail: [email protected] and studied the clinical effects of this supplementa-
FATTY ACIDS COMPOSITION OF PLASMA PHOSPHOLIPIDS AND TRIGLYCERIDES IN CHILDREN WITH CYSTIC FIBROSIS tion. The study included fourteen young subjects, aged
The reduced availability of EFA has been reported to alter pat- between 6 and 15 years, affected by cystic fibrosis,
terns of circulating and tissue esterified acids and may deter- with pancreatic insufficiency and heterozygotes or
mine profound changes in membrane fluidity24,25 and cell sig- homozygotes for the ∆F508 mutation. The subjects
were matched by age and randomly assigned to either
Alterations in these processes may be particularly important an oil mixture supplemented (OM) group (n = 7), or to
for cells responding to chronic lung infections and for pan- a control (C) group (n = 7). In contrast to the control
creatic insufficiency that are frequently found in CF patients.
group, the patients with supplemented diet achieved
A few years ago, when the clinical definition of CF was first significant increases of the relative amount of C18:1 in
introduced, average survival did not exceed the pediatric age.
the triglycerides as well as a significant decrease in sat-
Nowadays, many CF patients survive until an adult age due to urated fatty acids (C 16:0, C 17:0, C 18:0, C 22:0).
ever advancing dietary management and therapeutical tech- Moreover, the ratio between LA acid and AA signifi-
cantly increased in the triglycerides of the OM group.
Relatively recent improvements in the dietary management In the phospholipids of the OM group, the relative
of children affected by CF include the introduction of both amount of C 18:1 and of palmitic acid increased signif-
enteric-coated microsphere pancreatic enzyme preparations, icantly whereas the relative amount of the most impor-
and a diet with about 40% of energy from lipids.
tant polyunsaturated fatty acids (PUFA) decreased.
The effects of administration of lipids with different fatty acid These results show that oleic acid can be absorbed and
composition on EFA availability and on the production of incorporated into the plasma triglycerides of CF
prostaglandins 2 (PG ) and of leukotrienes B4 (LTB ) in CF patients receiving pancreatic enzymes, whereas poor
patients28,29 have been investigated, as well. Different lipid incorporation of LA occurs. Despite the reduction in
composition of dietary supplements have been investigated, the relative amounts of phospholipid PUFA, the sup-
but two kinds of strategies are often suggested by literature.
plemented subjects did not reported adverse effects
One tries to correct poor EFA pattern with supplements rich There were no significant differences between groups
in LA30. Some authors reported that patients with this kind of in the clinical indexes recorded (height, weight, BMI,
supplemented diets achieved significant increases of energy Schwachman-Kulczycki score and FEV 1s). The results
intake, weight for height, body fat, as well as LA in plasma of this study showed that the supplementation with a
phospholipids31,32. Other authors reported that poor EFA pat- mixture of extravirgin olive and soybean oil was safe
tern was not corrected or was difficult to correct because of in seven CF patients treated during a 2-months period
malabsorption or other metabolic deficiencies33,34.
and no negative clinical effects were evident. However,
The second dietary strategy aims at the suppression of proin- further clinical trials will be necessary in order to bet-
flammatory eicosanoids.This is generally achieved by provid- ter evaluate the consequence of the observed changes
ing a supplement of long chain polyunsaturated ω-3 fatty in plasma fatty acids composition in a longer testing
acids with fish oil35-39. The dietary management with fish oil resulted in increased eicosapentaenoic acid (EPA) and DHAin plasma and tissues and reduced plasma LTB , and may pro- vide some benefits with relatively few adverse effects, but Introduction
there is still insufficient evidence to recommend routine useof supplements of omega-3 fatty acids in people with cystic Cystic fibrosis (CF) is characterized by abnormal levels of essential fatty acids (EFA) in plasma phospholipids. Linoleic A different approach in the dietary treatment of CF could acid (LA) and docosahexaenoic acid (DHA) have been report- involves the use of extra virgin olive oil.The processing tech- ed to be lower and palmitoleic acid (POA) higher in CF nology used for extra virgin olive oil is a mechanical cold patients than healthy subjects1-7. Moreover, recent studies crushing without any refining or organic solvent extraction found that membrane-bound arachidonic acid (AA) levels step and preserves its peculiar content of natural phenolic increased, whereas docosahexaenoic acid levels decreased in compounds. Recent literature data showed that the daily use the pancreas, lungs, and ileum of CF knock-out mice8-9.These of extra virgin olive oil has irrefutably beneficial effects in defects have been attributed to low fat diet10,11, fat malab- healthy subjects. Its peculiar polyphenols showed protective sorption12-14, abnormal lipid turnover in cell membranes15, effects in different animal models of inflammation41 and pos- altered desaturase activity14,16,17, increased oxidation of fatty sess an array of potentially beneficial lipoxygenase-inhibitory, acids18, increased production of eicosanoids19,20, abnormality prostaglandin-sparing, and antioxidant properties42,43.
in Ca++ induced AA release21, defective control of EFA utiliza- Moreover, recent report suggest that enrichment of LDL with tion22 by cystic fibrosis transmembrane regulation protein olive oil monounsaturated fatty acids reduces the lipoprotein (CFTR), and unpaired Cl- conductance23.
FATTY ACIDS, TOTAL POLYPHENOLIC COMPOUNDS AND VITAMIN E Olive and Soybean oil mixture
Compounds
a: the other alleles carried were N1303 (1 subjects), G85E (1 subject), G542X (1 subject); b: the other alleles carried were N1303 (2 subjects), G85E (1 subject) In the current study, the results of a new strategy aimed at the realization of a practical, low cost integrator, for daily use inthe dietary management of FC subjects, are reported. Weinvestigated the plasma phospholipids and triglycerides fatty and the types of food consumed.The general approach to the acids composition of CF patients subjected to a dietary sup- nutritional management of CF patients was a high-fat, high- plement constituted of a mixture of 50% extra virgin olive oil energy diet designed to achieve 120-150% of the recom- and 50% soybean oil and studied the clinical effects of this mended daily allowance (RDA). Lipid intake represented 30- 40% of total energy intake. The visible portion of dietarylipids of OM was substituted by dietary supplement, consti-tuted of a mixture of 50% extravirgin olive oil and 50% soy- Subjects
bean oil, in reason to achieve 8-10% of total energy intake.Themixture was fed to OM for 2 months. The fatty acids, total The study included fourteen young subjects, aged between 6 polyphenolic compounds and vitamin E content of the oil and 15 years, affected by cystic fibrosis, with pancreatic insuf- mixture is reported in Table 2. Body weight, height and the ficiency and heterozygotes or homozygotes for the ∆F508 forced expiratory volume in 1 s (FEV ) of the subjects were mutation. The patients were recruited from the Pediatric recorded, using standard techniques at the beginning and at Division of “G. Salesi”Hospital (Ancona, Italy), matched by age the end of the dietary supplemented period.
and randomly assigned to either an oil mixture supplement-ed (OM) group (n = 7), or to a control (C) group (n = 7).Themain subjects characteristics are presented in Table 1.
Therapies prescribed to patients before the experimentalperiod were not suspended.All the subjects required regular L-α-phosphatidylcholine dipalmitoyl (PC), pancreatic enzyme supplementation and received antibiotics phatidylethanolamine dipalmitoyl (PE), L-α-phosphatidylinos- therapy when exacerbation of their pulmonary disease itol ammonium salt from bovine liver (PI), N-palmitoyl-D- occurred. Four subjects of the OM and three of the C groups sphingomyelin (Sph) and L-α-lysophosphatidylcholine from received recombinant human deoxyribonuclease I egg yolk (L-PC) were purchased from Sigma Chemicals Co.
(Pulmozyme). Two subjects of the OM and three of the C groups were taking antileukotrienes (Montelukast). Subjects Pre-coated silica gel plates were purchased from Merck receiving insulin, corticosteroids and other drugs affecting fat (Darmstadt, Germany). HPLC grade solvents were purchased mass were excluded, as well as patients without ∆F508 allele from BDH Ltd (Poole, UK). All other chemicals, with noted or with chronic intestinal pseudobstruction, renal insuffi- exceptions, were obtained from Sigma Chemicals Co. (St.
ciency, abnormal liver function or any metabolic disorder.
Plasma collection and lipid extraction Blood samples were obtained by venopuncture. Clotting wasprevented with EDTA and the plasma fraction collected by Dietary intake was assessed by using a 7-d food (household centrifugation at 1500 g for 20 min at 4°C. Plasma samples measures) diary. Subjects were given instructions and format- were stored at -20°C for lipid analysis. Total lipids were ted recording sheets to document the timing, the quantity extracted from plasma according to the method of Folch45.
FATTY ACIDS COMPOSITION OF PLASMA PHOSPHOLIPIDS AND TRIGLYCERIDES IN CHILDREN WITH CYSTIC FIBROSIS Two drops of 1:1 (v/v) aqueous hydrochloric acid were After extraction with n-hexane, the methyl esters were concen- added to 500 mg of serum. The acidified serum was vigor- trated under nitrogen. The analysis was performed with a ously mixed with 10 ml of chloroform/methanol (2:1, v/v); Chrompack (Middelburg, The Netherlands) CP-9003 gas chro- the solution was filtered under vacuum and 2 ml of 0.88% matograph, on a SP 2340 (30 m x 0.32 mm i.d., 0,20 µm film KCl were added.The chloroform layer was recovered and the thickness) column (Supelco, Bellefonte, CA), equipped with an solvent was evaporated under nitrogen.The lipid extract was on-column temperature programmed injection system (TP-OCI) redissolved in 500 ml of chloroform/methanol/water (5:5:1, and flame-ionization detector (FID).The carrier gas was helium and the oven temperature was maintained at 60°C for 3 min,then raised to 220°C in 55 min. The TP-OCI temperature wasmaintained at 60°C for 6 min, then raised to 220°C in 8 min.
Peak identification was carried out by comparing retentiontimes (RTs) with those of pure standards provided by Sigma Phospholipid classes were resolved by high-performance liq- Chemicals Co. (St. Louis, MO) and Supelchem Inc. (Bellefonte, uid chromatography (HPLC) on a Hypersil Si (150 mm x 4.6 PA), and by comparison with the results published in litera- mm i.d., 3 µm particle size) column (Phenomenex,Torrance, USA). The separation was obtained with a gradient elutionstarting at 100% A, decreasing to 0% A in 10 min, held at 0% Afor 15 min and then back to 100% A in 5 min. The mobile Oil mixture analysis
phase A was CHCl /MeOH/NH (80:19.5:0.5, v/v), and the mobile phase B was CHCl /MeOH/H O/NH (60:34:5.5:0.5, The crude oil was saponified according to the procedures v/v).The flow rate was 0.8 ml/min.The HPLC system consist- detailed in “Norme Grassi e Derivati” (Method NGD C 12- ed of a degassing unit (Gastorr GT-103), a ternary gradient 1976)49. The vitamin E content was detected in the module (Jasco LG-980-02), a pump module (Jasco PU-980), unsaponifiable fraction by gas chromatography using a Carlo and a light scattering detector (Polymer Labs PL-EMD 960).
Erba (Milano, Italy) HRGC 5160 Mega instrument in the con- Peak identification was carried out by comparing retention ditions reported by Frega et al.50.The fatty acid composition times with those of pure standards provided by Sigma was determined in the saponifiable matter, in the condition reported above, following the treatment with diazomethane Calibration curves for quantitative HPLC analyses were run (CH N )51 to convert free fatty acids into their methyl ester.
with the commercial standards of PC, L-PC, PI, PE, and Sph.
Total polyphenolic compounds were detected via the spec- Standard solution contained 0.2-1 mg ∗ml-1 for Sph, 0.06-0.28 trofotometric procedure reported by Montedoro et al.52.
mg ∗ml-1 for PE, 0.1-0.4 mg ∗ml-1 for PI, 0.02-0.1 mg ∗ml-1 forL-PC, and 0.4-1 mg ∗ml-1 for PC, and were injected with anincreasing concentration order in each run. Three replicates Statistical analysis
were run for each concentration. Regression analyses weredone with a linear function; r2 varied between 0.988 for PC to Results are expressed as mean ± standard deviation (SD). All variables were recorded at inclusion (day 0) and after twomonths (day 60). Data were compared with respect to time(day 60 versus day 0) and between groups (OM versus con- Fatty acid analysis
trol group) by using the Student’s t test.
Phospholipids and triglycerides were separated by thin layerchromatography (TLC) using pre-coated silica gel G plates (20x20x0.25 cm) and n-hexane/diethylether (60:40, v/v) asdeveloping solvent.
Data on height, weight, puberty status, clinical status and lung The separated lipid classes were detected under UV light function were recorded. BMI distribution of subjects partici- after nebulization with 1% solution of 2’,7’-dichlorofluo- pating in the present study were categorized in the ideal rescin in ethanol.The plates were scraped off and lipids were range for age. Schwachman-Kulczycki score and FEV 1s sug- extracted from silica: the triglyceride band was recovered gested that the CF subjects were not too severely affected.
with diethylether and phospholipids were recovered with Mean BMI and FEV 1s of OM group and C group recorded at chloroform/methanol/water (5:5:1, v/v/v).
the beginning and at the end of the experimental period are Fatty acid methyl esters were prepared from phospholipids reported in Table 3. There were no significant differences and triglycerides by acid-catalyzed transmethylation accord- between groups in the clinical indexes recorded.There were no significant differences on plasma phospholipids composi- CLINICAL INDEXES OF THE PATIENTS SUPPLEMENTED WITH OIL MIXTURE EFFECT OF DIETARY SUPPLEMENT ON PLASMA PHOSPHOLIPIS Phospholipids
Mean ± SD
Mean ± SD
(µg/g plasma)*
1231.8±128.3 1293.9±148.4 1195.4±160.3 1118.3± 207.7 OM: oil mixture group; C: control group; *: mean ± SD OM: oil mixture group; C: control group; BMI: Body Mass Index; FEV 1s: forced expi-ratory volume in 1s; a: possible score range from 0 to 100, the lowest being theworst; b: days of antibiotic therapy prescribed to patients from the beginning to the The relative amount of palmitic acid also increased signifi- cantly from 28.5 ± 2.2 to 31.0 ± 1.9 and the relative amountof the most important PUFA decreased significantly: AA and tion as reported in Table 4.There were significant differences dihomo-γ-linolenic acid (C 20:3 ω6) decreased respectively between groups in the fatty acid composition of plasma from 3. 5 ± 0.7 to 2.8 ± 0.6 and from 8.6 ± 1.1 to 6.5 ± 1.2; triglycerides (Table 5) and phospholipids (Table 6). In con- DHA decreased from 2.2 ± 0.7 to 1.6 ± 0.4.
trast to the control group, the patients with supplementeddiet achieved significant increases of the relative amount ofC18:1 in the triglycerides (respectively from 40.3 ± 5.5 to Discussion
51.7 ± 9.7 in the OM group, and from 43.9 ± 4.9 to 41.6 ± 5.1in the C group), as well as a significant decrease in saturated The objective of this 2-months study was to evaluate, in chil- fatty acids (C 16:0, C 17:0, C 18:0, C 22:0). Moreover, the ratio dren affected by CF, aged between 6 and 15 years, the clinical between LA acid and AA significantly increased in the triglyc- effects and the impact on the plasma fatty acid composition of a dietary supplement consisting of a mixture of 50% extravirgin In the phospholipids of the OM group, the relative amount of olive oil and 50% soybean oil.The mixture was high in monoun- C 18:1 increased significantly from 13.4 ± 1.5 to16.4 ± 2.3.
saturated fatty acids, especially in oleic acid (OA) and LA.
EFFECT OF DIETARY SUPPLEMENT ON FATTY ACID COMPOSITION OF PLASMA TRIGLYCERIDES Fatty acids (%)a
OM: oil mixture group; C: control group; P: Student’s test value (NS, P > 0,05); a: mean ± SD of the relative amount; b: OM versus C groups; c: OM at day 60 versus OM at day FATTY ACIDS COMPOSITION OF PLASMA PHOSPHOLIPIDS AND TRIGLYCERIDES IN CHILDREN WITH CYSTIC FIBROSIS EFFECT OF DIETARY SUPPLEMENT ON FATTY ACIDS COMPOSITION OF PLASMA PHOSPHOLIPIDS Fatty acids (%)a
OM: oil mixture group; C: control group; P: Student’s test value (NS, P > 0,05); a: mean ± SD of the relative amount; b: OM versus C groups; c: OM at day 60 versus OM atday 0 After two months of dietary treatment, the relative amount of ratio between ω6 and ω3 fatty acids remained unchanged. No all C 18:1 isomers increased in the triglycerides of the sup- complications resulted from administration of the oil mixture plemented subjects, particularly at the expanse of saturated to these CF patients. In addition, the antibiotic therapy in fatty acids. However the relative amount of LA remained occurrence of respiratory exacerbation episodes was shorter unchanged despite the high LA content of the dietary sup- in the OM group with respect to the control group, in most plementation (Table 2).These results showed that oleic acid can be easily absorbed and incorporated into the plasma The results of this study showed that the supplementation triglycerides of CF patients receiving pancreatic enzymes, with a mixture of extravirgin olive and soybean oil was safe whereas the intestinal uptake of LA could be impaired. In in seven CF patients treated during a 2-months period and no fact, the ratio between C 18:1 and LA in plasma triglycerides negative clinical effects were evident. However, further clini- of the OM group was 3.7 ± 1,5, at the inclusion, and 5.9 ± 1,5, cal trials will be necessary in order to better evaluate the con- at the end of the experimental period, whereas it was only sequence of the observed changes in plasma fatty acids com- 1.9 in the mixture. This could be attributed to impaired position in a longer testing period. It will be a major chal- intestinal absorption or to abnormal metabolism of LA33,34.
lenge for the future to modify the oil mixture fatty acids com- Increased levels of C 18:1 were found also in plasma phos- position in order to achieve a better profile in the plasma fat- pholipid fatty acids, particularly at the expanse of PUFA.
ty acids of CF patients and to investigate changes in the fatty Because the essential fatty acids status in the plasma of CF acids composition of CFTR regulated tissues.
patients is often compromised by deficiencies in PUFA, aworsening of the disease could have been expected from thereduction in the relative amounts of phospholipid PUFA Acknowledgements
recorded in this study. However, the supplemented subjectsdid not reported adverse effects despite this reduction in the The authors wish to thank Dr. E. Boselli for the useful discus- relative amounts of PUFA in the phospholipids. Notably, the cell membranes of cystic fibrosis patients (in vitro study).
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Source: http://www.massimorenzi.net/sites/default/files/Fat.ac.FC.pdf