Association of Cystic Fibrosis with Abnormalities in Fatty Acid Metabolism
Steven D. Freedman, M.D., Ph.D., Paola G. Blanco, M.D., Munir M. Zaman, M.D., Julie C. Shea, B.A., Mario Ollero, D.V.M., Ph.D., Isabel K. Hopper, R.N., Deborah A. Weed, R.N., Andres Gelrud, M.D., Meredith M. Regan, Sc.D., Michael Laposata, M.D., Ph.D., Juan G. Alvarez, M.D., Ph.D., and Brian P. O'Sullivan, M.D.
Background Patients with cystic fibrosis have altered levelsof plasma fatty acids. We previously demonstrated that arachidonicacid levels are increased and docosahexaenoic acid levels aredecreased in affected tissues from cystic fibrosis–knockoutmice. In this study we determined whether humans with mutationsin the cystic fibrosis transmembrane conductance regulator (CFTR)gene have a similar fatty acid defect in tissues expressingCFTR.
Methods Fatty acids from nasal- and rectal-biopsy specimens,nasal epithelial scrapings, and plasma were analyzed from 38subjects with cystic fibrosis and compared with results in 13obligate heterozygotes, 24 healthy controls, 11 subjects withinflammatory bowel disease, 9 subjects with upper respiratorytract infection, and 16 subjects with asthma.
Results The ratio of arachidonic to docosahexaenoic acid wasincreased in mucosal and submucosal nasal-biopsy specimens (P<0.001)and rectal-biopsy specimens (P=0.009) from subjects with cysticfibrosis and pancreatic sufficiency and subjects with cysticfibrosis and pancreatic insufficiency, as compared with valuesin healthy control subjects. In nasal tissue, this change reflectedan increase in arachidonic acid levels and a decrease in docosahexaenoicacid levels. In cells from nasal mucosa, the ratio of arachidonicto docosahexaenoic acid was increased in subjects with cysticfibrosis (P<0.001), as compared with healthy controls, withvalues in obligate heterozygotes intermediate between thesetwo groups (P<0.001). The ratio was not increased in subjectswith inflammatory bowel disease. Subjects with asthma and thosewith upper respiratory tract infection had values intermediatebetween those in subjects with cystic fibrosis and those inhealthy control subjects.
Conclusions These data indicate that alterations in fatty acidssimilar to those in cystic fibrosis–knockout mice arepresent in CFTR-expressing tissue from subjects with cysticfibrosis.
The mechanisms by which mutations in the gene encoding the cysticfibrosis transmembrane conductance regulator (CFTR) proteinlead to the expression of cystic fibrosis are unclear. Explanationsmust take into account the increased viscosity of ductal fluidsas well as the excessive host inflammatory response.1,2,3,4,5,6Alterations in the metabolism of fatty acids may have an importantrole.7 A deficiency of essential fatty acids has been describedin patients with cystic fibrosis8 that is characterized by adecrease in the plasma levels of linoleic acid (18:2n–6)and docosahexaenoic acid (22:6n–3) with a compensatoryincrease in the levels of eicosatrienoic acid (20:3n–9)9,10,11(Figure 1). Similar changes have been observed in well-nourishedpatients with cystic fibrosis14,15 and obligate heterozygotesfor mutant CFTR,16 suggesting that this abnormality is due notto nutritional deficiencies but to CFTR dysfunction.14 The resultsof studies of fatty acids in erythrocytes from patients withcystic fibrosis have been inconsistent.12,17 Therefore, we examinedwhether alterations in arachidonic acid (20:4n–6), docosahexaenoicacid, and other fatty acids are present in CFTR-regulated tissuesfrom patients with cystic fibrosis and whether such abnormalitiesare specific to cystic fibrosis or are present in other inflammatorydisorders.
Figure 1. An Overview of the n–3, n–6, and n–9 Biosynthetic Pathways.
In the n–3 pathway, the essential fatty acid -linolenic acid is converted to eicosapentaenoic acid and docosahexaenoic acid. In the n–6 pathway, the essential fatty acid linoleic acid is converted to arachidonic acid. Eicosatrienoic acid is formed in the n–9 pathway from oleic acid. Levels of some fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, can be affected by exogenous intake. Modified from Biggemann et al.12 and Sprecher.13
Methods
Enrollment of Subjects
The study was conducted from October 2000 to September 2003.All subjects provided written informed consent before enrollment.The institutional review board at the University of MassachusettsMedical School (Worcester) and Beth Israel Deaconess MedicalCenter (Boston) approved the protocols. Subjects with cysticfibrosis met the consensus-statement requirements for the diagnosisof this disease18 and were followed at the UMass Memorial CysticFibrosis Center (Worcester). A total of 38 subjects with cysticfibrosis were enrolled (31 with pancreatic insufficiency and7 with pancreatic sufficiency), all of whom had pulmonary disease.Pancreatic insufficiency was defined by the requirement forexogenous pancreatic enzymes for the treatment of clinicallydiagnosed steatorrhea. Forced expiratory volume in one secondas a percentage of predicted values, body-mass index (the weightin kilograms divided by the square of the height in meters),and the National Institutes of Health prognostic score, as definedby Taussig et al.,19 were obtained. All subjects were encouragedto eat high-fat (35 to 40 percent of calories), high-caloriediets and had regular visits with a nutritionist, in accordancewith the guidelines of the Cystic Fibrosis Foundation.20
We also evaluated five groups of control subjects. We studied13 obligate heterozygotes: 12 were the mothers of subjects whowere homozygous for the F508 CFTR mutation, and 1 was a fatherwith an identified F508 mutation. We studied 11 subjects withinflammatory bowel disease (8 with Crohn's disease and 3 withulcerative colitis), which was diagnosed endoscopically in theproper clinical context of chronic diarrhea and the exclusionof other causes. The diagnosis of acute upper respiratory tractinfection in nine control subjects, which was presumed to beviral in nature, was based on the presence of a self-limitedacute pharyngitis and rhinitis in the absence of an identifiedbacterial cause in otherwise healthy subjects. Twenty-five controlsubjects with physician-diagnosed asthma were enrolled. Twenty-threeused daily inhaled corticosteroid therapy to control their symptoms,and the other two used beta-agonists. Blood from all 25 subjectswith asthma was sent to Ambry Genetics for CFTR gene analysis.All CFTR exons, at least 20 bases 5' and 3' into each intron,as well as select portions of introns associated with specificmutations, were analyzed. Only subjects with asthma who hadno known CFTR mutations (other than V470) were included in theanalysis. A total of 24 healthy controls were recruited at bothsites after a review of their medical history. Exclusion criteriafor all control subjects included a family history of cysticfibrosis or features consistent with the presence of cysticfibrosis (chronic lung disease, male infertility, chronic sinusitis,and chronic pancreatitis) or the use of drugs that affect fattyacids (systemic corticosteroids, isotretinoin, and ursodiol).
Tissue Procurement
A single physician obtained nasal-biopsy specimens (approximately3 mm) from the inferior turbinate after the submucosal injectionof 1 percent lidocaine. The inferior turbinate was chosen becausethis is where CFTR function is directly examined in patientswith cystic fibrosis by means of testing of nasal potentialdifference.21 Rectal tissue, which also expresses CFTR,22,23was obtained with the use of a suction-biopsy catheter placed5 cm from the anal verge. All biopsy specimens were immediatelyplaced into a glass vial containing 1.2 ml of chloroform–methanol(2:1 vol/vol) containing 30 µl of heptadecanoic acid (1mg per milliliter) (17:0) in chloroform–methanol (1:1vol/vol) as an internal standard. No complications occurred.
Scrapings of nasal mucosa from the inferior turbinate were obtainedwith a plastic Rhinoprobe (Arlington Scientific). A 2-cm rowof cells was scraped from the inferior turbinate as describedpreviously for the purification of CFTR messenger RNA,24 andthis procedure was repeated five to eight times to obtain sufficienttissue. Scrapings were placed in 10 ml of sterile normal salineand centrifuged at 400xg for five minutes at room temperature;the cell pellet was washed once and then processed for fatty-acidanalysis after extraction with chloroform–methanol.
Blood and Lipid Analysis
Peripheral venous blood was obtained from subjects at the timeof biopsy or nasal mucosal scraping, collected in a heparin-treatedtube, and centrifuged at 400xg for 15 minutes at room temperature.The plasma was then removed, and the lipids were extracted bythe addition of chloroform–methanol (2:1 vol/vol). Thelipid samples were then vortexed, sonicated, and centrifugedat 400xg.25 The organic phase of each sample was removed, andfatty acids were methylated.25 Fatty acid methyl esters werequantified by gas chromatography–mass spectrometry withthe use of a gas chromatograph (HP5890 Series II, Hewlett–Packard)equipped with a Supelcowax SP-10 capillary column (Supelco)attached to a mass spectrometer (HP-5971, Hewlett–Packard).Total ion monitoring was performed, encompassing mass rangesfrom 50 to 550 atomic mass units. The mass of fatty acid methylesters was determined by comparing areas of unknown fatty acidmethyl esters with a fixed concentration of internal standard(equivalent to 30 µg of heptadecanoic acid for nasal-biopsyand rectal-biopsy specimens and 15 µg for nasal mucosascrapings and plasma). All specimens were analyzed in a blindedfashion. In addition, samples from different groups of subjectswere mixed in with the assay batches.
Statistical Analysis
Demographic characteristics are summarized as means (±SD)and compared between groups with the use of analysis of variance.Data on fatty acids are summarized as medians and interquartileranges. Exclusions based on lipid analyses were made beforestatistical analyses were performed. The levels of fatty acidsin nasal- and rectal-biopsy specimens were compared among threegroups of subjects with use of the nonparametric Jonckheere–Terpstratest26 for a priori ordered groups. The fatty acids from nasal-mucosascrapings and plasma lipids were compared among groups withthe use of Kruskal–Wallis tests for unordered groups.If the result of the global test was statistically significant,then Wilcoxon rank-sum tests were used to compare groups ina pairwise fashion and exact two-sided P values were reported.The analysis used SAS software, version 8.0 (SAS Institute),and StatXact software, version 5 (Cytel Software).
Results
Fatty Acids in Nasal-Biopsy and Rectal-Biopsy Specimens
The demographic characteristics of the subjects with cysticfibrosis and the healthy control subjects who underwent a biopsyare shown in Table 1. No subject had a weight loss of more than5 percent in the month before biopsy collection. For technicalreasons, data could not be analyzed from one subject with cysticfibrosis and pancreatic insufficiency and four healthy controlsubjects who underwent nasal biopsy and from three subjectswith cystic fibrosis and pancreatic insufficiency and two healthycontrol subjects who underwent rectal biopsy.
Table 1. Demographic Characteristics of Subjects Who Underwent Rectal and Nasal Biopsies.
Table 2 shows the mole percentages of each fatty acid in nasal-biopsyspecimens. There were significant differences between subjectswith cystic fibrosis and pancreatic insufficiency and healthycontrol subjects only for linoleic acid (P=0.01), arachidonicacid (P=0.008), and docosahexaenoic acid (P=0.01).
Table 2. Complete Fatty Acid Analysis of Nasal-Biopsy Tissues from Subjects with Cystic Fibrosis and Pancreatic Insufficiency and Healthy Control Subjects.
Since the biologic effects of fatty acids are dependent notonly on the absolute levels of a particular fatty acid but alsoon the ratio of n–6 to n–3 fatty acids,28Figure 2shows the individual data points for arachidonic acid anddocosahexaenoic acid, as well as for the ratio of arachidonicto docosahexaenoic acid. Arachidonic acid levels were increased,with reciprocal changes in docosahexaenoic acid, in subjectswith cystic fibrosis and pancreatic insufficiency, as comparedwith healthy control subjects (Figure 2). The median ratio ofarachidonic to docosahexaenoic acid in the subjects with cysticfibrosis was 13.0, as compared with 5.8 in healthy control subjects(P<0.001). No overlap in the individual data points was observed.Although approximately 25 percent of the subjects with cysticfibrosis were having an acute exacerbation of their pulmonarydisease at the time of tissue procurement, the range of valuesfor the ratio of arachidonic to docosahexaenoic acid was similarin subjects with an acute pulmonary flare (median, 12.3; range,9.0 to 13.0) and those without a flare (median, 13.7; range,11.2 to 16.3). Similarly, there was no significant differencein these values when male subjects (median, 13.0; range, 12.3to 16.3) were compared with female subjects (median, 12.4; range,9.0 to 14.4).
Figure 2. Levels of Arachidonic Acid and Docosahexaenoic Acid and the Ratio of Arachidonic to Docosahexaenoic Acid in Nasal-Biopsy Specimens from Nine Subjects with Cystic Fibrosis and Pancreatic Insufficiency, Seven Subjects with Cystic Fibrosis and Pancreatic Sufficiency, and Seven Healthy Control Subjects.
Data analysis was not possible for one subject with cystic fibrosis and pancreatic insufficiency and four healthy control subjects. Horizontal lines indicate the median values. There were significant differences among the three groups in the levels of arachidonic acid (P=0.008) and docosahexaenoic acid (P=0.002) and in the ratio of arachidonic to docosahexaenoic acid (P<0.001) with the use of the Jonckheere–Terpstra test. The group of healthy control subjects had a significantly lower ratio than the subjects with cystic fibrosis and pancreatic sufficiency (P=0.02) and the subjects with cystic fibrosis and pancreatic insufficiency (P<0.001) with use of the Wilcoxon rank-sum test.
Subjects with cystic fibrosis and pancreatic sufficiency werealso studied, since they may have unique fatty acid profilesowing either to less severe CFTR dysfunction than subjects withpancreatic insufficiency29 or to improved nutritional status.The median ratio of arachidonic to docosahexaenoic acid in subjectswith cystic fibrosis and pancreatic sufficiency was 10.7, whichwas intermediate between the values in subjects with cysticfibrosis and pancreatic insufficiency and healthy control subjects(P<0.001 by the Jonckheere–Terpstra test).
In rectal tissue, the median docosahexaenoic acid level waslower (P=0.05) in subjects with cystic fibrosis and pancreaticinsufficiency than in healthy controls, with no significantdifferences in arachidonic acid levels (Figure 3). The medianratio of arachidonic to docosahexaenoic acid of 12.0 in rectal-biopsyspecimens from subjects with cystic fibrosis and pancreaticinsufficiency was similar to the ratio in nasal mucosal biopsyspecimens and was significantly higher than the median valueof 5.6 in healthy controls (P=0.02). There was no significantdifference between subjects with pancreatic sufficiency andthose with pancreatic insufficiency.
Figure 3. Levels of Arachidonic Acid and Docosahexaenoic Acid and the Ratio of Arachidonic to Docosahexaenoic Acid in Rectal-Biopsy Specimens from Seven Subjects with Cystic Fibrosis and Pancreatic Insufficiency, Seven Subjects with Cystic Fibrosis and Pancreatic Sufficiency, and Nine Healthy Control Subjects.
Data analysis was not possible for three subjects with cystic fibrosis and pancreatic insufficiency and two healthy control subjects. Horizontal lines indicate the median values. There were significant differences among the three groups in the levels of docosahexaenoic acid (P=0.01) and in the ratio of arachidonic to docosahexaenoic acid (P=0.009) but not in the levels of arachidonic acid (P=0.17) with the use of the Jonckheere–Terpstra test. The ratio in the group of healthy control subjects differed significantly from the ratio in the group with cystic fibrosis and pancreatic sufficiency (P=0.01) and in the group with cystic fibrosis and pancreatic insufficiency (P=0.02) with use of the Wilcoxon rank-sum test.
Fatty Acid Levels in Scrapings of Nasal Epithelia Mucosa
The demographic characteristics of the subjects from whom nasalepithelial scrapings were obtained are shown in Table 3. Theindividual data points for arachidonic and docosahexaenoic acidsand the ratio of these two fatty acids are shown in Figure 4.Docosahexaenoic acid levels in nasal mucosal scrapings weresimilar to those in nasal-biopsy specimens. The range of arachidonicacid values was larger in nasal mucosal scrapings than in biopsysamples. The median ratio of arachidonic to docosahexaenoicacid in nasal mucosal cells was 9.1 in subjects with cysticfibrosis and pancreatic insufficiency and 5.5 in obligate heterozygotes,and values in both groups were significantly different fromthe median value of 3.2 in healthy controls (P<0.001 foreach comparison).
Figure 4. Levels of Arachidonic Acid and Docosahexaenoic Acid and the Ratio of Arachidonic to Docosahexaenoic Acid in Nasal Mucosal Scrapings from 21 Subjects with Cystic Fibrosis and Pancreatic Insufficiency, 13 Obligate Heterozygotes, 16 Healthy Control Subjects, 11 Subjects with Inflammatory Bowel Disease (IBD), 9 Subjects with Upper Respiratory Tract Infection (URI), and 16 Subjects with Asthma.
Horizontal lines indicate the median values. Overall, levels of arachidonic acid (P=0.01) and docosahexaenoic acid (P<0.001) and the ratios of arachidonic to docosahexaenoic acid (P<0.001) differed significantly among the six groups of subjects when analyzed by means of the Kruskall–Wallis test.
Other inflammatory diseases were also examined. Eight of the11 subjects with inflammatory bowel disease (7 with Crohn'sdisease and 1 with ulcerative colitis) had mild to moderatelyactive disease requiring therapy, with a weight loss of no morethan 5 percent in the month before sample collection. The medianratio of arachidonic to docosahexaenoic acid was lower in subjectswith inflammatory bowel disease than in healthy control subjectsand subjects with cystic fibrosis (P=0.03 and P<0.001, respectively)(Figure 4). This difference was mostly due to the increase indocosahexaenoic acid levels.
Upper respiratory tract infection in otherwise healthy subjectswas associated with a greater range of values, with the medianratio of arachidonic to docosahexaenoic acid intermediate betweenthat observed in subjects with cystic fibrosis (P<0.001)and healthy control subjects. As a control for chronic airwayinflammation, nasal scrapings from 25 subjects with asthma wereobtained. Nine of these subjects were excluded from the analysisowing to the presence of known CFTR mutations, novel variantsof unknown functional significance, or inadequate samples. Ofthe remaining 16 subjects, 11 (69 percent) had two copies ofthe V470 polymorphism, 4 had one copy (25 percent), and 1 hadno copies (6 percent). In total, 26 of 32 alleles (81 percent)had the V470 variant. As shown in Figure 4, arachidonic acidlevels were higher in the subjects with asthma than in the subjectswith cystic fibrosis (P=0.01) and healthy control subjects (P=0.04),whereas levels of docosahexaenoic acid were intermediate betweenthese two groups (P<0.001 for the comparison of healthy controlsand subjects with cystic fibrosis). The median ratio of arachidonicto docosahexaenoic acid in subjects with asthma was 6.9, whichwas intermediate between that in subjects with cystic fibrosis(9.1, P<0.001) and healthy control subjects (3.2, P<0.001).
Fatty Acid Profiles in Plasma
The fatty acid profile in plasma was examined to determine whetherthere were alterations in the levels of fatty acids similarto those in the tissues (Table 4). Linoleic acid and eicosatrienoicacid were also examined to determine whether an essential fattyacid–like deficiency was present in our subjects, likethat described in other reports.9,10,11 Docosahexaenoic acidlevels were significantly lower in the plasma of subjects withcystic fibrosis than in healthy control subjects (P<0.001),with intermediate values observed for obligate heterozygotes(P<0.001 for the comparison with subjects with cystic fibrosisand P=0.13 for the comparison with healthy control subjects).In contrast, arachidonic acid levels did not differ significantlyamong the groups (P=0.56). There was no significant differencein linoleic acid or eicosatrienoic acid levels between subjectswith cystic fibrosis and healthy control subjects.
Our data demonstrate an altered ratio of arachidonic to docosahexaenoicacid in CFTR-expressing tissues in subjects with cystic fibrosis.The biologic effects of fatty acids depend not only on the absolutelevels of a particular fatty acid but also on the ratio of n–6to n–3 fatty acids.28 The magnitude of these alterationsand the fatty acids involved are similar to our earlier resultsin cystic fibrosis–knockout mice.30 These data suggestthat CFTR may have a role in cellular fatty acid metabolismand that abnormalities in fatty acids may affect the cysticfibrosis phenotype.
To determine whether acute or chronic inflammation can modifyfatty acids, we studied three control groups with inflammatorydisease. Inflammatory bowel disease was associated with verylow ratios of arachidonic to docosahexaenoic acid. Subjectswith asthma and acute upper respiratory tract infection hada median value intermediate between that in subjects with cysticfibrosis and healthy control subjects. On the basis of the findingof an increased prevalence of CFTR gene mutations in subjectswith asthma,31 we performed CFTR sequencing in all subjectswith asthma, and those with mutations or variants were excluded.A large proportion of our subjects with asthma had the M470Vallele, which may cause decreased CFTR function.32 The fattyacid abnormality in this group could be due to CFTR dysfunction,and such a possibility would be supported by the finding thatobligate heterozygotes have fatty acid changes of similar magnitude.However, the fact that subjects with acute upper respiratorytract infection had fatty acid levels similar to those of thesubjects with asthma suggests that inflammation may also explainthis degree of fatty acid abnormality. The much greater changein the levels in subjects with cystic fibrosis indicates thatinflammation alone is unlikely to explain all the changes inthe arachidonic and docosahexaenoic acid levels.
Two lines of evidence support the suggestion that this fattyacid abnormality is caused by abnormal or mutant CFTR and notby abnormal intestinal absorption of fat due to pancreatic insufficiency.First, the ratio of arachidonic to docosahexaenoic acid wasabnormal in nasal and rectal tissue from subjects with cysticfibrosis and pancreatic sufficiency, who have normal assimilationof micronutrients and macronutrients. Second, the ratio in nasalmucosal scrapings from obligate heterozygotes was intermediatebetween values in subjects with cystic fibrosis and healthycontrol subjects. This finding is in agreement with previousreports that CFTR function is not normal in obligate heterozygotes33,34and is associated with the development of cystic fibrosis–relateddiseases in humans such as sinusitis35 and chronic pancreatitis.36,37
The abnormal tissue levels of docosahexaenoic acid cannot solelybe due to the low plasma levels, since fatty acid metabolismis cell-specific and the fatty acids in cells of CFTR-regulatedtissues may not reflect the levels of fatty acids in the circulation.38,39,40,41The findings in cystic fibrosis–knockout mice that thefatty acid abnormality is present only in affected tissues andthat plasma fatty acids were the same as those in their wild-typelittermates30 indicates that the fatty acid defect can occurin the absence of alterations in plasma fatty acids. Althoughfuture studies should strictly control the dietary intake offatty acids, especially eicosapentaenoic acid and docosahexaenoicacid, diet alone does not fully explain the fatty acid abnormalitywe identified.
Although the mechanism by which CFTR regulates fatty acid metabolismis unknown, the low docosahexaenoic acid levels may be importantin the excessive host inflammatory response in cystic fibrosis.Docosahexaenoic acid is converted to docosatrienes and 17S seriesresolvins, which are potent antiinflammatory mediators thatare normally generated during the resolution of inflammation.42,43Therefore, the low docosahexaenoic acid levels in patients withcystic fibrosis may explain, at least in part, the inflammatorystate associated with this disease and may have important therapeuticimplications. We have previously shown that oral administrationof high doses of docosahexaenoic acid to cystic fibrosis–knockoutmice corrects the fatty acid abnormality, reverses the histologicchanges in the pancreas and ileum, and decreases neutrophillevels in mice with pseudomonas lipopolysaccharide–inducedpneumonia.30,44 It will be important to determine whether correctionof this fatty acid defect represents a treatment for cysticfibrosis.
Funded by grants from the Cystic Fibrosis Foundation and GenzymeCorporation (to Drs. Freedman and Alvarez) and supported inpart by a grant (RR 01032, to the Beth Israel Deaconess MedicalCenter General Clinical Research Center) from the National Institutesof Health.
We are indebted to Joanne Cluette-Brown for her analysis ofthe lipid samples by gas chromatography–mass spectrometry;to Maynard Hansen, M.D. (Ear, Nose, and Throat), and StephenHardy, M.D. (Pediatric Gastroenterology), for procuring nasal-and rectal-biopsy samples, respectively; and to Rhoda Spaulding,M.S.N., F.N.P., for the enrollment of subjects at UMass MemorialHealth Care.
Source Information
From the Departments of Medicine (S.D.F., P.G.B., M.M.Z., J.C.S., M.O., I.K.H., D.A.W., A.G., M.M.R.) and Obstetrics and Gynecology (M.O.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston; the Division of Laboratory Medicine, Massachusetts General Hospital and Harvard Medical School, Boston (M.L.); Instituto de Infertilidad Masculina, Centro de Infertilidad Masculina Androgen, Hospital San Rafael, La Coruña, Spain (J.G.A.); and the Department of Pediatrics, UMass Memorial Health Care, University of Massachusetts Medical School, Worcester (B.P.O.).
Address reprint requests to Dr. Freedman at Beth Israel Deaconess Medical Center, Dana 552, 330 Brookline Ave., Boston, MA 02215, or at sfreedma{at}caregroup.harvard.edu.
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F508 CFTR mutation, and 1 was a father with an identified 
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-linolenic acid is converted to eicosapentaenoic acid and docosahexaenoic acid. In the n–6 pathway, the essential fatty acid linoleic acid is converted to arachidonic acid. Eicosatrienoic acid is formed in the n–9 pathway from oleic acid. Levels of some fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, can be affected by exogenous intake. Modified from Biggemann et al.
