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Originally published In Press as doi:10.1194/jlr.M400238-JLR200 on October 1, 2004

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Journal of Lipid Research, Vol. 45, 2277-2286, December 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

No indications for altered essential fatty acid metabolism in two murine models for cystic fibrosis

Anniek Werner*, Marloes E. J. Bongers*, Marcel J. Bijvelds{dagger}, Hugo R. de Jonge{dagger} and Henkjan J. Verkade1,*

* Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, Academic Hospital Groningen, The Netherlands
{dagger} Department of Biochemistry, Erasmus Medical Center, Rotterdam, The Netherlands

Published, JLR Papers in Press, October 1, 2004. DOI 10.1194/jlr.M400238-JLR200

1 To whom correspondence should be addressed. e-mail: h.j.verkade{at}med.rug.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A deficiency of essential fatty acids (EFA) is frequently described in cystic fibrosis (CF), but whether this is a primary consequence of altered EFA metabolism or a secondary phenomenon is unclear. It was suggested that defective long-chain polyunsaturated fatty acid (LCPUFA) synthesis contributes to the CF phenotype. To establish whether cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction affects LCPUFA synthesis, we quantified EFA metabolism in cftr–/–CAM and cftr+/+CAM mice. Effects of intestinal phenotype, diet, age, and genetic background on EFA status were evaluated in cftr–/–CAM mice, {Delta}F508/{Delta}F508 mice, and littermate controls. EFA metabolism was measured by 13C stable isotope methodology in vivo. EFA status was determined by gas chromatography in tissues of cftr–/–CAM mice, {Delta}F508/{Delta}F508 mice, littermate controls, and C57Bl/6 wild types fed chow or liquid diet. After enteral administration of [13C]EFA, arachidonic acid (AA) and docosahexaenoic acid (DHA) were equally 13C-enriched in cftr–/–CAM and cftr+/+CAM mice, indicating similar EFA elongation/desaturation rates. LA, ALA, AA, and DHA concentrations were equal in pancreas, lung, and jejunum of chow-fed cftr–/–CAM and {Delta}F508/{Delta}F508 mice and controls. LCPUFA levels were also equal in liquid diet-weaned cftr–/–CAM mice and littermate controls, but consistently higher than in age- and diet-matched C57Bl/6 wild types. We conclude that cftr–/–CAM mice adequately absorb and metabolize EFA, indicating that CFTR dysfunction does not impair LCPUFA synthesis.

A membrane EFA imbalance is not inextricably linked to the CF genotype. EFA status in murine CF models is strongly determined by genetic background.

Abbreviations: AA, arachidonic acid, 20:4n-6; ALA, {alpha}-linolenic acid, 18:3n-3; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; DHA, docosahexaenoic acid, 22:6n-3; EFA, essential fatty acid; KHB, Krebs-Henseleit buffer; LA, linoleic acid, 18:2n-6; LCPUFA, long-chain polyunsaturated fatty acid; PL, phospholipids

Supplementary key words arachidonic acid • cystic fibrosis transmembrane conductance regulator • docosahexaenoic acid • essential fatty acid deficiency • {alpha}-linolenic acid • linoleic acid • modifier genes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A deficiency of essential fatty acids (EFA) or their long-chain polyunsaturated metabolites (LCPUFA) has frequently been reported in cystic fibrosis (CF) patients (1–4) and has formerly been attributed to fat malabsorption due to pancreatic insufficiency. Current high-fat, hypercaloric nutritional strategies and improved pancreas enzyme replacement therapies can usually maintain patients in optimal nutritional status, thus normalizing EFA status in many CF patients (5). Nonetheless, several reports still indicate the occurrence of EFA deficiency in CF (6–8). Although some authors have suggested that residual fat malabsorption and increased EFA turnover in CF may compromise EFA status (9, 10), the exact pathophysiology of EFA deficiency in CF patients has not been elucidated.

A direct link between cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction and EFA metabolism has been postulated by Gilljam et al. (11), and Bhura-Bandali et al. (12) described impaired EFA incorporation into phospholipids (PL) in human pancreatic CF cells. In cftr–/–UNC mice, Freedman et al. (13) reported a profound membrane fatty acid imbalance, characterized by increased concentrations of arachidonic acid (AA) and decreased concentrations of docosahexaenoic acid (DHA) in membrane PL of organs typically affected in CF, such as pancreas, lung, and intestine. Oral supplementation with DHA, but not with its precursor ALA, corrected this lipid imbalance and was reported to reverse certain pathological features of the disease. These studies suggested that CFTR exerts control over LCPUFA synthesis from EFA, and that impaired EFA processing primarily contributes to CF pathology (13, 14). However, it has not been elucidated whether perturbed EFA status in CF is a primary result of CFTR malfunction or secondary to fat malabsorption or increased turnover.

Several CF mouse models have been developed in the past decade, including total null mice with no detectable CFTR production (15–17), and mice with the {Delta}F508 mutation, with low-level residual CFTR activity (18). Similar to CF patients, CF mouse models display significant phenotypic variability, particularly concerning the severity of gastrointestinal symptoms such as intestinal obstruction and fat malabsorption.

To assess the effect of CFTR on LCPUFA synthesis, we quantified conversion of EFA into LCPUFA in vivo in cftr–/–CAM mice and littermate controls. In addition, we analyzed fecal fatty acid excretion and membrane fatty acid composition in tissues of cftr–/–CAM mice (University of Cambridge) (17) and homozygous {Delta}F508 mice (18), as well as of their respective littermate controls. These particular CF models have been shown to differ in intestinal phenotype, with fat malabsorption present in cftr–/–CAM mice but absent in {Delta}F508/{Delta}F508 mice (19). Furthermore, we determined the effects of age, diet, and genetic background on EFA status in these mouse models and in C57Bl/6 wild-type mice. Our results indicate that cftr–/–CAM mice adequately absorb, elongate, and desaturate intragastrically administered EFA, and that a membrane fatty acid imbalance in CF-affected tissues is not inherent to the CF genotype in mouse models with and without fat malabsorption. Rather, EFA status in CF mice is strongly determined by genetic background, diet, and age.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
C57Bl/6/129 cftr–/–tm1CAM mice and cftr+/+tm1CAM littermates (17), homozygous {Delta}F508 mice and sex-matched littermate controls (N/N) of FVB/129 background (18) and wild-type C57Bl/6 mice were accommodated at the breeding colony at the Erasmus Medical Center, Rotterdam, The Netherlands. Southern blotting of tail-clip DNA was performed to verify the genotype of individual animals (20). Mice were housed in a light-controlled (lights on 6 AM to 6 PM) and temperature-controlled (21°C) facility and were allowed tap water and standard laboratory chow (Hope Farms BV Woerden, The Netherlands) or liquid diet (Peptamen) ad libitum from the time of weaning. The Ethical Committee for Animal Experiments in Rotterdam approved the experimental protocols.

Experimental diets
The standard laboratory chow contained 6 weight% fat and 14 energy% fat, and had the following fatty acid composition: 18.2 mol% palmitic acid (C16:0), 7.0 mol% stearic acid (C18:0), 25.8 mol% oleic acid (C18:1n-9), 39.1 mol% linoleic acid (C18:2n-6), 3.5 mol% {alpha}-linolenic acid, 0.3 mol% arachidonic acid, and 0.05 mol% docosahexaenoic acid (Hope Farms BV, Woerden, the Netherlands). The Peptamen liquid diet (Nestle Clinical Nutrition, Brussels, Belgium) contained 3.7 g fat/100 ml (33 energy%) and had the following fatty acid composition: 16.4 mol% palmitic acid (C16:0), 6.7 mol% stearic acid (C18:0), 22.2 mol% oleic acid (C18:1n-9), 43.6 mol% linoleic acid (C18:2n-6), 4.6 mol% {alpha}-linolenic acid, 0.1 mol% arachidonic acid, and 0.08 mol% docosahexaenoic acid.

Experimental procedures
The cftr–/–CAM mice and cftr+/+CAM littermates (n = 5–6 per group) were fed standard laboratory chow from weaning. At 3 months of age, mice were anesthetized with isoflurane and a baseline blood sample was obtained by tail bleeding. Subsequently, a 100 µl lipid bolus containing uniformly labeled [13C]LA and [13C]ALA was slowly administered by intragastric gavage, for determination of in vivo conversion of EFA into LCPUFA and partitioning to different organs. The lipid bolus was composed of olive oil mixed with [U-13C]LA (0.40 mg) and [U-13C]ALA (0.40 mg) (Martek Biosciences, Columbia, MD). [U-13C]LA and [U-13C]ALA were 99% 13C-enriched, with a chemical purity exceeding 97%. At 24 h after bolus administration, a large blood sample was obtained by cardiac puncture and pancreas, liver, lungs and intestine were removed and immediately stored at –80°C until further analysis. Intestine and lungs were flushed with ice-cold 0.9% (w/v) NaCl solution before storage. Blood was collected in heparinized vials and plasma and erythrocytes were separated by centrifugation. Erythrocyte membrane lipids were hydrolyzed and methylated for fatty acid analysis the same day (21) to prevent fatty acid oxidation, and plasma was stored at –80°C.

To establish the effect of fat malabsorption, diet, age, and genetic background on body EFA status, homozygous {Delta}F508 mice of FVB/129 background, sex-matched N/N littermates, and cftr–/–CAM and cftr+/+CAM mice (n = 5–6 per group) were fed standard laboratory chow from weaning. At the age of 3 months, mice were anesthetized with isoflurane and sacrificed by means of cardiac puncture. Lung, pancreas, and jejunum were removed and samples of each were immediately stored at –80°C for fatty acid and protein analysis. Fecal fatty acid excretion was quantified by gas chromatographic analysis of feces aliquots obtained after a 72 h fat balance.

A separate group of cftr–/–CAM and cftr+/+CAM mice and C57Bl/6 wild types (n = 6 per group) were weaned at 23 days of age, and subsequently put on Peptamen liquid diet ad libitum for 7 days. At postnatal day 30, mice were anesthetized and blood, pancreas, lung, jejunum, ileum, and liver samples were obtained. Jejunum, ileum, and lungs were flushed with ice-cold saline and ileal mucosa was separated from submucosal layers by scraping with a glass microscope slide onto an ice-cooled glass plate. Pancreatic cell suspensions were prepared by mechanical dissociation and addition of collagenase as described by Bruzzone et al. (22). Lung tissue was flushed with Krebs-Henseleit buffer (KHB), pH 7.4, containing 0.5% BSA to rinse off contaminating blood. Lung tissue was then finely cut and suspended in 10 ml of oxygenated KHB containing 1,000 units of collagenase, 2,000 units of DNase, and 0.5 units of thermolysin and was incubated for 30 min at 37°C. The lung cell suspension was then sedimented through KHB containing 4% BSA and washed once in KHB. All organ samples were stored at –80°C until further analysis.

Analytical techniques
13C enrichment analysis Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS; DeltaPlusXL, Thermo Finnigan, Bremen, Germany) was used to measure 13C enrichment of LA and ALA and their metabolites. The GC-C-IRMS was equipped with a 50 m x 0.22 mm BPX70 capillary column and the injector temperature was set at 275°C with splitless injection. The gas chromatograph oven was programmed from an initial temperature of 50°C to a final temperature of 250°C in 3 steps (50°C, held 1 min isotherm; 50–100°C, ramp 7°C/min; 100–225°C, ramp 10°C/min; 225–250°C, ramp 25°C/min, held 10 min). Helium was used as a carrier gas, with a constant flow rate of 0.5 ml per minute. The 12CO2+ and 13CO2+ ions were measured at m/z 44 and 45, respectively. Correction for 17O was achieved by measurement of 18O abundance at m/z 46.

Fatty acid analysis Fatty acid profiles were determined by hydrolyzing, methylating, and extracting total plasma lipids and erythrocyte membrane lipids as described by Muskiet et al. (21). For fatty acid analysis of liver, intestine, pancreas, and lung tissue, samples were mechanically homogenized in 0.9% NaCl solution and lipids were extracted from aliquots of tissue homogenate as described by Bligh and Dyer (23). The lipid extract was partly methylated in toto for GC analysis, and partly fractionated into PL, cholesterol esters, triacylglycerols, diacylglycerols, monoacylglycerols, and free fatty acids using TLC (20 x 20 cm, Silica gel 60 F254, Merck) with hexane/diethyl ether/acetic acid (80:20:1, v/v/v) as solvent. TLC plates were dried and colored by iodine, and PL and triacylglycerol spots were scraped. Of these scrapings, fatty acid methyl esters were prepared as mentioned above. To account for losses during lipid extraction, heptadecaenoic acid (C17:0) was added to all samples as internal standard prior to Bligh and Dyer procedures (23). Butylated hydroxytoluene was added as antioxidant. Aliquots of chow diet and feces were freeze-dried and homogenized, after which lipids were hydrolyzed, methylated, and extracted for fatty acid analysis. Similarly, fatty acid composition of Peptamen liquid diet was determined after dissolution in chloroform/methanol (2:1 v/v).

Fatty acid methyl esters were separated and quantified by gas-liquid chromatography (GLC) on a Hewlett Packard gas chromatograph model 6890, equipped with a 50 m x 0.2 mm Ultra 1 capillary column (Hewlett Packard, Palo Alto, CA) and a flame ionization detector as described previously (24). We verified the purity of AA and DHA peaks as separated by GLC, using a gas chromatography-mass spectrometer (GC-MS; Finnigan MAT SSQ7000), equipped with either a 50 m x 0.2 mm Ultra 1 capillary column or a 50 m x 0.22 mm BPX70 capillary column (SGE, Weiterstadt, Germany). Both methyl esters and pentafluorobenzyl bromide (PFB-Br) derivatives of tissue fatty acids were analyzed, but no indications for impurity of AA or DHA peaks could be detected.

Protein analysis Total protein contents of tissue homogenates were determined with Folin phenol reagent as described by Lowry et al. (25). Standard Pierce BSA was used as reference.

Calculations
Abundance of 13C was expressed as {delta}13CPDB value (i.e., the difference between the sample value and baseline compared with Pee Dee belemnite limestone). The {delta}13CPDB values were converted to atom % 13C values. Enrichment (atom % excess) was calculated by subtracting baseline 13C abundance from all enriched values.

Relative concentrations (mol%) of individual fatty acids in plasma, erythrocytes, liver, intestine, pancreas, and lung were calculated by summation of all fatty acid peak areas and subsequent expression of areas of individual fatty acids as a percentage of this amount. Fatty acid contents were quantified by relating the areas of their chromatogram peaks to that of the internal standard heptadecaenoic acid (C17:0).

Statistics
All results are presented as means ± SD for the number of animals indicated. Data were statistically analyzed using Student's t-test or, for comparison of more than two groups, ANOVA test with post hoc Bonferroni correction. Statistical significance of differences between means was accepted at P < 0.05. Analyses were performed using SPSS for Windows software (SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vivo conversion of [13C]EFA into LCPUFA
LCPUFA in specific tissues originate either from the diet or from endogenous synthesis by elongation and desaturation of EFA. Because CFTR dysfunction has been postulated to affect EFA tissue incorporation or rate of metabolism (12, 13, 26, 27), we quantified in vivo the appearance in different organs of ingested 13C-labeled EFA and their conversion into LCPUFA. At 24 h after intragastric administration of [13C]LA and [13C]ALA, 13C enrichment of LA and ALA could be demonstrated in all analyzed tissues of cftr–/–CAM mice and cftr+/+CAM controls. [13C]LA and [13C]ALA concentrations were not significantly different between cftr–/–CAM and cftr+/+CAM mice (Fig. 1) . Similarly, [13C]AA levels did not significantly differ between cftr–/–CAM mice and littermate controls. 13C-enrichment of DHA was below detection limit in liver triacyclglycerols (data not shown) and in lung PL, but [13C]DHA in jejunum, pancreas, and liver PL was similar in cftr–/–CAM mice and controls. The ratios between [13C]LA and [13C]AA and between [13C]LA and [13C]DHA in liver, pancreas, lung, and intestine of cftr–/–CAM and cftr+/+CAM mice were highly comparable, suggesting adequate rates of LA and ALA elongation and desaturation in this CF mouse model.



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  		Fig. 1. 13C enrichment of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in phospholipids (PL) of pancreas, lung, intestine, and liver of cftr+/+ mice (gray bars) and cftr–/– mice (white bars) at 24 h after intragastric administration of [13C]LA and [13C]LA. Individual fatty acid 13C enrichment was calculated from the difference between the sample value and baseline compared with Pee Dee Belemnite limestone ({delta}13CPDB), and is expressed as 100 x atom % excess. Data represent means ± SD of six mice per group. No significant differences in 13C enrichment were detected between cftr+/+ and cftr–/– mice, indicating normal conversion of essential FAs (EFA) into long-chain PUFAs (LCPUFA).

 
Fatty acid composition of feces and tissue homogenates
To determine the effects of intestinal phenotype on EFA status, we analyzed fatty acid composition of feces and of CF-affected organs in CF mouse models with and without fat malabsorption. Figure 2 shows the daily fecal excretion of the main dietary fatty acids in {Delta}F508/{Delta}F508 and cftr–/–CAM mice and their respective controls. Fecal fatty acid excretion was similar in homozygous {Delta}F508 mice and controls, in contrast to cftr–/–CAM mice, which secreted significantly more fatty acids in feces than littermate controls, confirming the presence of fat malabsorption in this CF mouse model. Malabsorption of saturated fatty acids was slightly more pronounced than that of unsaturated fatty acids.



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  		Fig. 2. Relative concentrations of palmitic acid (PA, 16:0), stearic acid (SA, 18:0), oleic acid (OA, 18:1n-9), linoleic acid (LA, 18:2n-6), and docosahexaenoic acid (DHA, 22:6n-3) in fecal lipid extracts of homozygous {Delta}F508 mice and cftr–/– mice and their respective littermate controls. Fecal fat excretion was quantified by means of a 72 h fecal fat balance. Individual fatty acid concentrations are expressed as mmol of fatty acid excreted per day. Data represent means ± SD of 5–6 mice per group. No significant differences were detected for any of the fatty acids between homozygous {Delta}F508 mice and controls, but daily fecal fatty acid excretion was significantly higher in cftr–/– mice than in cftr+/+ controls (* P < 0.05).

 
Figure 3 shows LA, AA, ALA, and DHA concentrations in tissue homogenates of pancreas, lung, and jejunum in homozygous {Delta}F508 mice and cftr–/–CAM mice compared with their respective littermate controls. No significant differences were observed in relative concentrations of these EFA and LCPUFA, nor of saturated and nonessential fatty acids (data not shown).



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  		Fig. 3. Relative concentrations of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in total lipid extracts of homozygous {Delta}F508 mice and cftr–/– mice and their respective littermate controls. Individual fatty acid concentrations are expressed as mol% of total fatty acids. Data represent means ± SD of 5–6 mice per group. No significant differences were detected for any of the fatty acids between {Delta}F508 or cftr–/– mice and their littermate controls.

 
The fatty acid composition of the total lipid fraction depends on the fatty acid composition of the various lipid classes present (i.e., triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids, cholesterol esters, free fatty acids), and the proportions of these lipid classes may vary considerably between organs. Since PL may be a more adequate indicator for determination of EFA status, we specifically analyzed fatty acid composition of tissue PL fractions of CF-affected organs. Figure 4A shows relative fatty acid concentrations in the PL fraction of pancreas, lung, and jejunum of the two CF mouse models and their respective controls. DHA concentrations in pancreas PL were 25% lower in {Delta}F508/{Delta}F508 mice than in N/N controls (P < 0.05); in lung PL, there was a small significant increase of AA in {Delta}F508/{Delta}F508 compared with N/N mice (5.3 ± 0.7 vs. 4.3 ± 0.5, respectively; P < 0.05). In jejunum PL of {Delta}F508/{Delta}F508 mice, however, AA was 37% decreased (3.1 ± 1.3 vs. 5.0 ± 0.7, P < 0.05) and ALA was 15% increased (0.48 ± 0.04 vs. 0.41 ± 0.04, P < 0.01) compared with N/N controls. Similar differences were not observed in tissues of cftr–/–CAM mice, in which LA, AA, ALA, or DHA concentrations were consistently similar to those of cftr+/+CAM littermates.





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  		Fig. 4. A: Relative concentrations of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in purified PL extracts of homozygous {Delta}F508 mice and cftr–/– mice and their respective littermate controls. Individual fatty acid concentrations are expressed as mol% of total fatty acids. Data represent means ± SD of 5–6 mice per group, *P < 0.05. B: Relative concentrations (mol%) of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in pancreas PL related to body weight of homozygous {Delta}F508 mice (open circles) and cftr–/– mice (open squares) and their respective littermate controls (N/N, closed circles; cftr+/+, closed squares). No correlations were detected between body weight and individual fatty acid concentrations. Data represent means ± SD of 5–6 mice per group. C: Absolute concentrations (nmol) of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in pancreas PL expressed per milligram pancreas protein for homozygous {Delta}F508 mice and cftr–/– mice and their respective littermate controls. Individual fatty acid concentrations were quantified by relating the areas of their chromatogram peaks to that of the internal standard heptadecaenoic acid (C17:0). No significant differences in absolute fatty acid concentrations per milligram protein were detected between homozygous {Delta}F508 mice and cftr–/– mice and their respective littermate controls. Data represent means ± SD of 5–6 mice per group.

 
The severity of the CF phenotype has been implicated in the EFA status of CF patients (7). Body weight is an important clinical parameter related to severity of CF symptoms (28), exemplified by a consistently lower weight of cftr–/–CAM mice compared with littermate controls (26.5 ± 4.4 g vs. 30.7 ± 3.8 g, respectively, P < 0.05). In contrast, homozygous {Delta}F508 mice, displaying normal fat absorption as a consequence of milder gastrointestinal pathology, show normal weight gain (22.4 ± 1.9 g vs. 23.0 ± 1.4 g, NS). To investigate the possible influence of nutritional status on EFA levels, EFA molar percentages were related to body weight for each individual mouse (Fig. 4B). Neither in cftr–/–CAM nor in {Delta}F508/{Delta}F508 mice or their corresponding controls could a significant correlation between relative EFA or LCPUFA concentrations and body weight be identified in pancreas (Fig. 4B), lung, or intestinal PL (data not shown).

In addition to relative fatty acid concentrations, absolute concentrations in the different tissues were determined and expressed per milligram protein. Figure 4C shows that absolute LA, ALA, AA, and DHA contents were similar in pancreas tissue of the two CF mouse models and their controls. Similarly, absolute fatty acid concentrations in lung or jejunum were not significantly different between CF and control mice (data not shown).

Membrane EFA concentrations appeared unaffected in the presently used CF mouse models compared with littermate controls. However, in 1-month-old cftr–/–UNC mice weaned on a liquid diet (Peptamen), a profound membrane lipid imbalance was reported (13). This suggests that dietary composition, caloric intake, and/or age affect EFA status. To test this hypothesis, fatty acid profiles were determined in tissues of 1-month-old cftr–/–CAM and cftr+/+CAM mice, after weaning on Peptamen liquid diet for 7 days. Furthermore, to assess the role of genetic background, EFA profiles were determined in tissues of age-matched, liquid diet-weaned C57Bl/6 wild-type mice.

Analysis of fatty acid composition of chow pellets and Peptamen revealed relatively small differences in EFA and LCPUFA contents, with Peptamen containing less AA and more DHA than standard chow (Fig. 5) . Although Peptamen is used frequently in CF mouse models to prevent intestinal obstruction and to improve nutritional status, 1-month-old cftr–/–CAM mice weaned on Peptamen still had a significantly lower body mass than their cftr+/+CAM littermates (11.4 ± 2.2 g vs. 13.6 ± 1.1 g, P < 0.05).



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  		Fig. 5. Fatty acid composition of standard laboratory chow and Peptamen elemental liquid diet. Data represent means ± SD of triple aliquot analyses of each diet. *P < 0.05 for linoleic acid (C18:2n-6), arachidonic aid (C20:4n-6), eicosapentaenoic acid (C22:5n-3), and docosahexaenoic acid (C22:6n-3) and # P < 0.001 for oleic acid (C18:1n-9) and behenic acid (C22:0).

 
The PUFA concentrations in pancreas, lung, and jejunum of 1-month-old, Peptamen-weaned cftr–/–CAM and cftr+/+CAM mice significantly differed from those of adult chow-fed cftr–/–CAM and cftr+/+CAM mice (Fig. 6A ; age effect). At the same age, however, LA, ALA, AA, and DHA concentrations in pancreas, lung, intestine, and plasma PL were not significantly different between cftr–/–CAM mice and littermate controls, neither at the age of 1 month after Peptamen weaning (Fig. 6B), nor at adult age during chow-feeding. However, AA and DHA concentrations were consistently higher than in age- and diet-matched C57Bl/6 wild-type mice for all tissues studied (P < 0.01). Similarly, LA concentrations in pancreas, lung, and jejunum PL of cftr–/–CAM and cftr+/+CAM mice were significantly higher than in C57Bl/6 wild-type controls, but not significantly different between cftr–/–CAM and cftr+/+CAM mice. ALA levels were low in all tissues analyzed, and, although there was a tendency for lower ALA values in wild-type C57Bl/6 mice compared with cftr–/–CAM mice, this reached significance only in plasma and ileum (P < 0.05 each). Similarly, fatty acid analyses of erythrocyte and ileum PL revealed consistently different fatty acid concentrations in C57Bl/6 mice, compared with cftr–/–CAM and cftr+/+CAM mice (data not shown). Other PUFA of the n-3 and n-6 series (i.e., 20:5n-3, 22:5n-3, 22:4n-6) also differed only between C57Bl/6 mice and cftr–/–CAM and cftr+/+CAM mice, and not between cftr–/–CAM mice and cftr+/+CAM littermates (data not shown).




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  		Fig. 6. A: Relative concentrations of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in purified PL extracts of pancreas, lung, and intestine of 1-month-old Peptamen-fed cftr+/+ mice (light gray bars) and cftr–/– mice (white bars), adult chow-fed cftr+/+ mice (black bars) and cftr–/– mice (dark gray bars). Individual fatty acid concentrations are expressed as mol% of total fatty acids. Data represent means ± SD of 6 mice per group. *P < 0.05 for DHA of pancreas and jejunum PL, for LA of lung and jejunum PL and for AA of jejunum PL from Peptamen-fed cftr+/+ and cftr–/– mice compared with adult chow-fed cftr+/+ and cftr–/– mice. No significant differences in fatty acid concentrations were detected between cftr+/+ and cftr–/– littermates. B: Relative concentrations of linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6), {alpha}-linolenic acid (ALA, 18:3n-3), and docosahexaenoic acid (DHA, 22:6n-3) in purified PL extracts of pancreas, lung, and intestine of cftr+/+ mice (gray bars), cftr–/– mice (white bars) and wild-type C57/Bl/6/129 mice (black bars). All mice were weaned on Peptamen liquid diet from postnatal day 23 and fatty acid analyses were performed at postnatal day 30. Individual fatty acid concentrations are expressed as mol% of total fatty acids. Data represent means ± SD of 6 mice per group. *P < 0.05 for LA and DHA and **P < 0.005 for AA in pancreas PL of wild-type C57/Bl/6/129 mice compared with cftr+/+ and cftr–/– mice. # P < 0.001 for LA, AA and DHA in lung and intestinal PL of C57/Bl/6/129 mice compared with cftr+/+ and cftr–/– mice. No significant differences in fatty acid concentrations were detected between cftr+/+ and cftr–/– mice.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We aimed to establish whether perturbed EFA metabolism and altered membrane EFA composition in CF-affected organs are inextricably linked to CF. Our present study in two murine models for CF shows no disturbance in either EFA metabolism or membrane fatty acid composition, indicating that a membrane EFA imbalance is not an intrinsic characteristic of the CF genotype in mice. By inference, our data indicate that the altered EFA compositions reported in CF are a secondary phenomenon, possibly related to inflammation or malnutrition.

Freedman et al. (13) reported markedly increased membrane AA and decreased DHA concentrations in CF-affected organs of a subset of cftr–/–UNC mice compared with nonlittermate C57Bl/6 controls. Oral supplementation with DHA, but not with its precursor ALA, corrected this membrane EFA imbalance and was reported to alleviate certain phenotypic manifestations of the disease. The authors suggested a causative relation between impaired capacity for conversion of EFA into LCPUFA and CF symptoms. Using [13C]EFA, we quantified rates of EFA elongation, desaturation, and tissue incorporation in vivo in cftr–/–CAM and cftr+/+CAM mice. After intragastric administration of [13C]EFA, levels of [13C]AA and [13C]DHA in jejunum, pancreas, and liver PL were equal in cftr–/–CAM and cftr+/+CAM mice, indicating that, in this CF mouse model, EFA elongation and desaturation is unaffected and that impaired LCPUFA synthesis is not an inextricable feature of the CF phenotype.

The phenotypic manifestations of CF are highly variable in both patients and different murine models for CF. We analyzed EFA status in two CF mouse models: homozygous {Delta}F508 mice with the {Delta}F508 exon 10 insertional mutation (18), expressing a mild phenotype without fat malabsorption, and cftr–/–CAM mice, in which exon 10 replacement results in complete absence of CFTR activity and a severe gastrointestinal phenotype, including fat malabsorption (17). For comparison, we used sex-matched littermates as controls. Quantification of fecal fatty acid excretion showed that cftr–/–CAM mice indeed malabsorbed dietary fatty acids. In neither of the two murine CF models, however, did we find indications for major membrane EFA alterations in CF-affected organs, compared with littermate controls. The slight and inconsistent alterations of AA levels that we did measure in lung and jejunum, and the marginally decreased DHA levels in pancreas, were found only in {Delta}F508/{Delta}F508 mice and not in cftr–/–CAM mice, despite the fact that the more severe phenotype of cftr–/–CAM mice would be expected to correlate with a higher incidence of membrane lipid imbalances (29). Only when cftr–/–CAM and cftr+/+CAM mice were compared with wild-type controls of different (C57Bl/6) genetic background did pronounced differences in membrane fatty acid composition become apparent.

The discrepancies between our observations and those of Freedman et al. (13) are unlikely to be explained by differences in preparative steps prior to GC injection. The discrepancies could, however, be related to the age difference between mice in our study (3 months) and in their study (1 month). In contrast to the chow-fed adult mice that we used, their 1-month-old liquid-diet weaned cftr–/–UNC mice displayed a profound lipid imbalance in CF-affected tissues, compared with C57Bl/6 mice. Theoretically, a conditional essentiality of dietary LCPUFA during early life may result in transiently low LCPUFA levels in young mice, which may resolve when EFA metabolizing capacity reaches maturity at adult age. Young cftr–/– mice might be more vulnerable than wild-type controls for such a transient deficiency of LCPUFA, due to impaired fat absorption in CF. However, comparison of membrane fatty acids of 1-month-old, liquid diet-fed cftr+/+CAM and cftr–/–CAM mice with those of 3-month-old, chow-fed mice indicated that the former actually had higher relative levels of EFA and LCPUFA. Similar to the 3-month-old mice, no differences in fatty acid composition were detected between 1-month-old cftr–/–CAM mice and cftr+/+CAM littermates, suggesting that differences in fatty acid levels between 1- and 3-month-old mice is more likely related to the different diets, or to an age-dependent effect unrelated to CFTR malfunction.

The different diets fed to cftr–/–UNC mice and cftr–/–CAM mice could theoretically account for the inconsistency regarding EFA levels in these two models. Both cftr–/–CAM mice and cftr–/–UNC mice display a severe phenotype characterized by fat malabsorption, goblet cell hyperplasia and failure to thrive, although cftr–/–UNC mice are more severely affected. When weaned on a chow-based diet, mortality due to intestinal obstruction is considerable in cftr–/–UNC mice during the first weeks of life. Weaning on a complete elemental liquid diet, such as Peptamen, significantly improves survival rates, but CF mice fed Peptamen remain considerably smaller than with normal littermates. To meet daily caloric needs, adult mice have to consume up to 15 ml of Peptamen per day (28), and lower intake may result in malnutrition. Striking similarities have been described between Peptamen-fed cftr–/–UNC mice and a malnourished CF mouse model regarding pulmonary cytokine profiles (30), suggesting that malnutrition secondary to liquid diet feeding may contribute to symptoms in Peptamen-fed CF mice (29). Relative EFA concentrations differ only slightly between chow and Peptamen, with Peptamen containing relatively less AA and more DHA than solid chow. Cftr–/–UNC mice fed Peptamen, however, had high levels of AA and low levels of DHA, which makes the fatty acid composition of the liquid diet an unlikely contributor to the observed membrane EFA imbalance in these mice. However, quantitative absorption studies would be required to fully exclude differences in net enteral uptake of EFA from chow or from Peptamen.

The discrepancy between our results and those of Freedman et al. (13) may also be due to variations inherent to the use of different mouse models for CF. To date, over 10 different murine CF models have been characterized; these can be categorized into mutants in which CFTR expression is simply disrupted [i.e., cftr–/–1HGU, cftr–/–HSC, cftr–/–BAY cftr–/–UNC and cftr–/–CAM mice (15–17, 31, 32)] and mutants that model specific clinical mutations, such as the {Delta}F508 mutation in cftr–/–EUR and cftr–/–1KTH mice (18, 33). Within the group with CFTR gene disruption, the potential to produce CFTR mRNA ranges from no detectable CFTR mRNA in absolute null mice (cftr–/–UNC, cftr–/–CAM, cftr–/–HSC) to mutants in which up to 10% of CFTR mRNA production is retained (cftr–/–1HGU). Generally, mice with lowest residual CFTR activity display the most severe phenotype, but phenotypic differences can also result from the different genetic backgrounds into which CFTR mutations have been introduced. The UNC mutation has been crossed into three different strains (i.e., C57Bl/6/129, B6D2/129 and BALB/C/129 mice), and the CAM mutation has been outcrossed to a C57Bl/6/129 population. Whereas we used sex-matched littermates as controls for cftr–/–CAM mice to evaluate EFA status, Freedman et al. (13) used nonlittermate, wild-type C57Bl/6 mice. Our present data indicate that genetic background and age have an overriding effect on EFA status in general and on DHA and AA levels in particular, so any meaningful comparisons of EFA status between CF mice and controls should take these confounding factors into account.

In addition to the specific type of CFTR mutation and to environmental influences, phenotypic variability between CF patients and mouse models is thought to be related to independently segregating disease-modifying genes. Proteins encoded by genes other than the CFTR gene may partially substitute for mutant CFTR, and individual variability in levels of tissue expression and functional activity for these other proteins may explain the interindividual phenotypic differences between patients or mice with identical CFTR mutations (34, 35). Several candidate modifier genes have been postulated to account for the wide spectrum of lung disease severity in patients homozygous for the {Delta}F508 mutation (36). Rozmahel et al. (32) demonstrated in mice the presence of a CFTR-independent locus that modulated severity of gastrointestinal disease, and Zielenski et al. (37) identified a similar modifier gene for meconium ileus on human chromosome 19. Similarly, the expression of liver disease has been described to be modulated by independently inherited modifier genes. This again underscores the prerequisite of using littermate controls in murine models for CF.

Our findings of normal membrane fatty acid composition in two CF mouse models correspond to results described by Dombrowsky et al. (38), who found normal levels of DHA and even decreased levels of AA in PL species of standard diet-fed adult cftr–/–1HGU mice. As in our study, the differences in EFA levels were very small, and inconsistent between PL classes. The fact that HGU mice have 10% residual CFTR mRNA makes conclusions regarding the role of CFTR in EFA metabolism difficult; nonetheless, both Dombrowsky's and our results underscore the variability in membrane PL composition between different CF mouse models. Strandvik et al. (7) described essential fatty acid deficiency in plasma PL of CF patients, but differences were small and AA levels were normal in all patients. The most pronounced EFA alterations were found in patients with severe mutations (i.e., {Delta}F508 and 394delTT), and, although no correlations were reported with other genotypes, a relation with fat malabsorption cannot be excluded.

In summary, from in vivo analyses of LCPUFA synthesis in a mouse model for CF, we conclude that impaired LCPUFA synthesis or imbalanced membrane fatty acid composition are not inextricable features of the CF phenotype. Fat malabsorption does not have a strong effect on EFA status in CF mice. Extrapolating these conclusions to CF patients could imply that sufficient oral EFA intake may effectively prevent compromised EFA status in CF. For studying essential fatty acid metabolism in murine CF models and inferring observations to the human condition, meticulous verification of mouse background strains and the use of littermate controls is crucial.


   ACKNOWLEDGMENTS
 
The authors would like to thank Rick Havinga, Henk Elzinga, and Ingrid Martini for their technical expertise and assistance in the experiments described in this article and Dr. Frans Stellaard for his help with the stable isotope studies. This study was supported by the Netherlands Organization for Scientific Research (NWO Grant 90462210).

Manuscript received June 21, 2004 and in revised form September 16, 2004.


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