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Potential Utility Of Plasma Fatty Acid[1] Potential Utility Of Plasma Fatty Acid[1] Document Transcript

  • Papers in Press. Published November 27, 2006 as doi:10.1373/clinchem.2006.077008 The latest version is at http://www.clinchem.org/cgi/doi/10.1373/clinchem.2006.077008 Clinical Chemistry 53:1 Lipids, Lipoproteins, 000 – 000 (2007) and Cardiovascular Risk Factors Potential Utility of Plasma Fatty Acid Analysis in the Diagnosis of Cystic Fibrosis Ibrahim Batal,1 Mhd-Bassel Ericsoussi,1 Joanne E. Cluette-Brown,1 Brian P. O’Sullivan,2 Steven D. Freedman,3 Juanito E. Savaille,1 and Michael Laposata1* Background: An altered distribution of fatty acids in Altered plasma and tissue concentrations of fatty acids in cystic fibrosis (CF)4 patients have been well described cells and tissues is found in patients with cystic fibrosis (1–7 ). Changes in concentrations of selected essential fatty (CF). In this study, we assessed the potential role of acids have been found in CF patients, notably decreases in plasma fatty acid analysis in the diagnosis of CF. the plasma and tissue concentrations of linoleic acid (18:2 Methods: In this 2-part study, we first used gas chroma- n-6) and docosahexaenoic acid (DHA; 22:6 n-3). Increases tography–mass spectrometry to analyze fatty acids in in the concentration of eicosatrienoic acid (Mead acid; 20:3 plasma from 13 CF patients and 11 controls without CF. n-9) have been identified in several studies (5–7 ). These We then used the fatty acid distribution data to identify alterations in fatty acid concentrations are significantly the fatty acids or multiple fatty-acid calculations most magnified in patients with severe variations in the CF effective in identifying CF patients. Part 2 of the study transmembrane conductance regulator (CFTR)5 gene, sug- was a blinded analysis of 10 CF patients and 9 controls gesting an association between the basic defect and ab- to directly test the effectiveness of the diagnostic param- normal essential fatty acid metabolism in CF patients (3 ). eters for CF identified from the plasma fatty acid It is unclear how CFTR variation leads to the dysregula- analysis. tion of fatty acid biosynthesis, but these fatty acid abnor- Results: In the nonblinded trial, the multiplication malities are clearly not attributable to pancreatic insuffi- product of (18:2 n-6) (22:6 n-3; each as percentage of ciency and malabsorption in CF patients (5–6 ). The total plasma fatty acid) was the most effective indicator metabolism of essential fatty acids involves a metabolic for distinguishing patients with CF from controls (P sequence of specific enzymes that desaturate, elongate, 0.0003). In part 2 (the blinded trial), this multiplication or shorten the fatty acids. Although the same enzymes are product was also the most effective indicator for distin- involved in both the n-3 and n-6 pathways, there is no guishing CF patients from controls (P 0.0008). interconversion of products between the 2 pathways. Conclusions: The product of (18:2 n-6) (22:6 n-3) is Thus, these essential fatty acids compete for the same effective for distinguishing CF patients from persons enzymes. A study with CF knockout mice demonstrated without CF. This diagnostic marker may have value as that arachidonic acid (20:4 n-6) concentrations are in- an alternative to the sweat chloride test in selected creased and DHA concentrations are decreased in tissues patients being evaluated for CF. and organs most affected in CF (8 ). Interestingly, oral © 2007 American Association for Clinical Chemistry administration of DHA was responsible for reversing the phenotypic alterations of CF in these mice (8 ). Alterations in fatty acids similar to those found in CF knockout mice were shown to be present in CFTR-expressing tissues 1 Division of Laboratory Medicine, Department of Pathology, Massachu- from human subjects with CF (1 ). setts General Hospital and Harvard Medical School, Boston, MA. 2 Department of Pediatrics, University of Massachusetts Memorial Health Care and University of Massachusetts Medical School, Worcester, MA. 3 Department of Medicine, Beth Israel Deaconess Medical Center and 4 Harvard Medical School, Boston, MA. Nonstandard abbreviations: CF, cystic fibrosis; DHA, docosahexaenoic *Address correspondence to this author at: Director of Clinical Laborato- acid; CFTR, cystic fibrosis transmembrane conductance regulator; FAME, ries, Massachusetts General Hospital, 235 Gray Bldg., 55 Fruit St., Boston, MA fatty acid methyl ester; GC-MS, chromatography-mass spectrometry; GCST, 02114. Fax 617-726-3256; e-mail mlaposata@partners.org. Gibson–Cooke Sweat Test. 5 Received July 27, 2006; accepted October 26, 2006. Human gene: CFTR, cystic fibrosis transmembrane conductance regula- tor (ATP-binding cassette subfamily C, member 7). Previously published online at DOI: 10.1373/clinchem.2006.077008 1 Copyright (C) 2006 by The American Association for Clinical Chemistry
  • 2 Batal et al.: Plasma Fatty Acids and CF Table 1. Demographic and genotypic characteristics of the CF patients. Nonblinded Trial Blinded Trial Sweat chloride, Sweat chloride, Sexa No. Age, years mEq/L Genotype No. Sex Age, years mEq/L Genotype 1 M 18 87 dF508/N1303K 1 F 18 114 dF508/dF508 2 M 18 91 dF508/dF508 2 M 21 115 dF508/dI507 Unknownb 3 F 28 117 dF508/dF508 3 M 22 dF508/dF508 4 F 17 88 dF508/dF508 4 F 17 101 dF508/dF508 dF508/3849 10kb C-Tc 5 M 18 84 dF508/G551D 5 F 12 35 6 F 41 150 dF508/dl507 6 F 7 101 dF508/dF508 NAd 7 M 15 100 dF508/W1282X 7 M 7 dF508/Y1092X 8 F 23 116 dF508/1717-1G3A 8 M 19 78 dF508/dF508 dF508/—e 9 F 16 NA 9 M 21 122 dF508/W1282X 10 F 23 NA dF508/dF508 10 F 18 104 dF508/dF508 11 M 19 129 dF508/G551D 12 F 19 115 dF508/G542X 13 M 32 NA dF508/— a M, male; F, female. b The patient had been seen at another medical center and was found to have a genotype consistent with CF; therefore, the sweat chloride test was not repeated in our study. c This genotype is consistent with negative sweat chloride test values in CF. d NA, not analyzed e —, Unidentified genotype. confirmed CF status (9–12 ). Another limitation for the Because certain alterations in plasma fatty acid compo- sweat test is that false-positive values may occur in sition in CF are highly reproducible, fatty acid analysis several clinical conditions readily distinguishable from CF may be useful as a diagnostic procedure for CF. The (13 ). In rare cases, some homozygous CF patients have altered fatty acid distributions that we observed in our sweat chloride concentrations within reference intervals if own studies with CF knockout mice and subsequently a second ameliorating or neutralizing variation in the with CF patients prompted us to reexamine our database CFTR gene product, such as R553Q, is also present (13 ). of CF patients to determine whether specific plasma fatty Nasal potential difference measurement is also used as acid changes can be used to differentiate between CF a diagnostic procedure in the evaluation for CF (14, 15 ). patients and persons not suffering from CF. An increased nasal potential difference is strong evidence The sweat chloride test is the gold standard screening for CF, but a value within reference intervals does not test for CF, but this test has limitations. A subset of exclude the diagnosis (16 ). False-negative results may patients with CF show values for the sweat chloride test arise, especially in the presence of a nasal polyp (17 ). To that are not diagnostic, and some CF patients demonstrate confirm a diagnosis of CF, an increased nasal potential a sweat chloride value within reference intervals despite Table 2. Mean (SD) plasma fatty acidsa of CF patients and healthy controls. Nonblinded Analysis Blinded Analysis b P valueb Fatty acid parameter CF patients (n 13) Controls (n 11) P value CF patients (n 10) Controls (n 9) (18:2 n-6) (22:6 n-3) 23.6 (2.7) 79.7 (10.5) 0.0003 20.2 (2.2) 42.4 (4.5) 0.0008 22:6 n-3 1.04 (0.14) 2.92 (0.56) 0.007 0.89 (0.06) 1.59 (0.2) 0.006 Total n-6/Total n-3 14.6 (1.13) 9.94 (1.23) 0.01 16.6 (0.5) 12.2 (1.3) 0.008 18:2 n-6 23.9 (1.7) 29.2 (1.9) 0.05 22.3 (1.2) 27.2 (1.1) 0.01 (22:5 n-6)/(22:6 n-3) 0.23 (0.03) 0.10 (0.02) 0.001 0.37 (0.03) 0.19 (0.03) 0.0008 (22:5 n-6)/(22:5 n-3) 0.47 (0.12) 0.38 (0.09) NS 1.05 (0.2) 0.65 (0.07) 0.04 (18:2 n-6) (22:6 n-3) (22:5 n-6) 97.6 (16.1) 321.9 (92.6) 0.04 67.4 (8.6) 194.7 (52.6) 0.04 (18:2 n-6)/(16:0) 1.08 (0.10) 1.41 (0.10) 0.03 0.93 (0.07) 1.11 (0.05) 0.05 (20:3 n-9)/(18:2 n-6) 0.02 (0.005) 0.002 (0.001) 0.01 0.006 (0.002) 0.001 (0.0006) NS (20:3 n-9) (22:5 n-6) 0.005 (0.002) 0.0004 (0.0002) 0.01 0.003 (0.001) 0.0002 (0.9) NS (18:2 n-6) (22:6 n-3) 20:3 n-9 0.34 (0.1) 0.05 (0.03) 0.01 0.12 (0.04) 0.03 (0.01) NS a Values mean (SE) for individual fatty acids are expressed as mole percentages, and calculations are derived from mole percentages of individual fatty acids. a Derived from the Student t-test. NS, not significant.
  • 3 Clinical Chemistry 53, No. 1, 2007 Fig. 1. The multiplication product of (18:2 n-6) (22:6 n-3; each as percentage of total plasma fatty acid) in the nonblinded analysis of 13 patients with CF and 11 non-CF controls and in the blinded analysis of Fig. 2. The multiplication product of (18:2 n-6) (22:6 n-3; each as 10 patients with CF and 9 non-CF controls. percentage of total plasma fatty acid) in CF patients and non-CF controls in the nonblinded trial, the blinded trial, and a study by The non-CF controls had significantly higher concentrations than the CF patients (P 0.0003 and 0.0008 in the nonblinded and blinded studies, respectively). Benabdeslam et al. (22 ). Data are presented as mean (SE) because the individual values were not available from Benabdeslam et al. (22 ). The non-CF controls had a significantly higher concentration than the CF patients in all studies. difference must be demonstrated twice. The nasal poten- tial difference measurement is a technically challenging controls [46 (11.2) years; range, 30 – 62 years]; patients and procedure (13 ). controls were selected after we reviewed medical records. These limitations of existing methods led us to pursue an alternative screening test involving plasma fatty acid plasma fatty acid isolation and methylation analysis to confirm the diagnosis of CF in patients with We obtained peripheral venous blood from nonfasting questionable sweat test results and positive clinical find- participants. Samples were collected in heparin-contain- ings for CF. ing vacuum tubes and centrifuged at 45g for 15 min at room temperature. Thereafter, the plasma was removed. Materials and Methods Fatty acids from plasma were isolated and methylated enrollment of patients according to Moser and Moser (20 ). The fatty acid methyl The nonblinded study was conducted from October ester (FAME) mixture was analyzed by gas chromatog- 2000 to September 2003, and the blinded study was raphy–mass spectrometry (GC-MS). conducted from September 2004 to August 2005. CF patients attending the University of Massachusetts Med- gc-ms fame identification and quantification ical Center and Beth Israel Deaconess Medical Center GC-MS analysis was performed on a Hewlett-Packard were included in the studies. Reference values were Series II 5890 gas chromatograph coupled to an HP-5971 obtained from healthy controls recruited at both sites. mass spectrometer (Agilent Technologies) equipped with Exclusion criteria for the controls included findings con- a Supelcowax SP-10 capillary column (Supelco). The oven sistent with the presence of CF, the use of drugs that affect temperature was maintained at 150 °C for 2 min, ramped fatty acid metabolism, or a family history of CF. All study at 10 °C/min to 200 °C and held for 4 min, ramped again patients had pancreatic insufficiency and met the criteria at 5 °C/min to 240 °C and held for 3 min, and then finally for CF diagnosis (18 ). All study participants provided ramped to 270 °C at 10 °C/min and maintained for 5 min. written informed consent before enrollment. Participants The injector and detector were maintained at 260 °C and were randomly selected for part 2 (blinded) of the study. 280 °C, respectively. Carrier gas flow rate was maintained In the nonblinded and blinded studies, the CF patients, at a constant 0.8 mL/min throughout. Total ion monitor- but not the controls, were encouraged to eat high-fat, ing was performed, encompassing mass ranges from 50 high-energy diets and to visit a nutritionist regularly, in to 550 atomic mass units. Peak identification was based on accordance with the guidelines of the Cystic Fibrosis comparison of both retention time and mass spectra of Foundation (19 ). The nonblinded study included 13 pa- the unknown peak to those of known standards within tients with CF [mean (SD) age, 22.1 (7.5) years; range, the GC-MS database library. A commercially available 15– 41 years] and 11 healthy controls [26.5 (7.2) years; standard mixture of FAMEs (Nucheck) was used to range, 19 –33 years]. The blinded study included 10 pa- calculate masses of fatty acids based on the response factor of 17:0 FAME (21 ). tients with CF [16.2 (5.6) years; range, 7–22 years] and 9
  • 4 Batal et al.: Plasma Fatty Acids and CF cutoff of 40 arbitrary units compared with 100%, 56%, Three samples from the control group were excluded 71%, and 100% for the blinded trial with the same cutoff from part 1 of the study because the fatty acid profile of 40 arbitrary units. Combined data from the blinded and indicated significant loss of fatty acids in the processing of nonblinded trials showed values for sensitivity, specific- the samples. In the excluded samples, the normally prom- ity, positive predictive value, and negative predictive inent fatty acids had areas that were 3–10-fold below the value of 96%, 75%, 81%, and 94%, respectively, for the area of the internal standard. Accepted samples were same cutoff of 40 arbitrary units. In addition, there were those in which the most prominent fatty acids were no technical limitations associated with sample collection. 3– 6-fold greater in peak area than the internal standard. The sensitivity of the assay for detection of CF was very In part 2 of the study, all specimens were analyzed in a high, and false positives were definitively identified as CF blinded fashion. or non-CF by genetic testing. statistical analysis Discussion Plasma fatty acid concentrations of CF patients and non-CF controls were compared. The unpaired Student Our data demonstrate that a multiplication product of t-test was used to evaluate differences between the means plasma (18:2 n-6) (22:6 n-3) can be used to differentiate of the 2 groups. Differences were considered statistically CF patients and non-CF controls in the majority of cases. significant at P 0.05. This multiplication product was the most effective param- eter in measuring plasma fatty acid status between CF Results patients and controls, and as a diagnostic marker, it provided a higher level of statistical significance than any The demographic and genotypic characteristics of the CF other mathematical operations or clinical markers tested. patients who were evaluated with plasma fatty acid In a study by Benabdeslam et al. (22 ), plasma phos- analysis are shown in Table 1. A genotype consistent with pholipid fatty acid analysis was performed with fasting the diagnosis of CF obviated the need for sweat testing in blood samples collected from 65 CF patients and 39 some patients. One of the CF patients from the blinded controls, whereas in our study, both the blinded and trial had a genotype that was consistent with a negative nonblinded trials were performed with samples collected sweat-chloride test value. from nonfasting CF patients and controls, a procedural Eleven different plasma fatty acid markers for differ- difference that may slightly alter plasma fatty acid com- entiating CF patients from controls, including (18:2 n-6) position. Other investigators have shown, however, that it (22:6 n-3), were tested in the nonblinded trial (Table 2). is unlikely that total plasma fatty acid composition is There were significant differences between CF patients significantly altered by a fasting period (23 ). The data in and controls for the multiplication product of (18:2 n-6) Fig. 2 show 3 paired comparisons between a CF group (22:6 n-3; P 0.0003), the ratio of (22:5 n-6):(22:6 n-3; P and a control group, including comparison of data from 0.001), and 22:6 n-3 alone (P 0.007). The multiplication total plasma fatty acid analysis in CF patients and controls product of (18:2 n-6) (22:6 n-3; each as percentage of using our data and data from the study by Benabdeslam total plasma fatty acid) most effectively distinguished et al. (22 ). The (18:2 n-6) (22:6 n-3) value separates CF patients with CF from controls (Fig. 1). patients from non-CF controls in all 3 paired comparisons. In the blinded trial, we tested 11 different plasma fatty The absolute values for (18:2 n-6) (22:6 n-3) are very acid markers (Table 2). Again, the multiplication product different, however, especially for the control groups. In of (18:2 n-6) (22:6 n-3) was the most statistically the current study, we used total plasma fatty acids be- significant parameter in distinguishing CF patients from cause samples do not require additional processing to controls (P 0.0008; Fig. 1). isolate phospholipids from total fatty acids. Thus, our The multiplication product of (18:2 n-6) (22:6 n-3) in method simplifies sample preparation for clinical use. In the nonblinded trial of our study showed sensitivity, specificity, positive predictive value, and negative predic- the 2 studies involving total fatty acids, although the tive value of 92%, 91%, 92%, and 91%, respectively, for a control groups were markedly different, the CF groups (22:6 n-3).a Table 3. Diagnostic sensitivity and specificity of sweat chloride test and of (18:2 n-6) Diagnostic Tests Technical Failure Rate Diagnostic Sensitivity Diagnostic Specificity Sweat test, % Mastella et al. (24 ) 3.6 91 100 Sweat test, % Warwick et al. (25 ) 14 93 99 (18:2 n-6) (22:6 n-3), % Negligible 92 91 Nonblinded analysis Blinded analysis, % (18:2 n-6) (22:6 n-3) Negligible 100 56 Blinded nonblinded analysis, % (18:2 n-6) Negligible 96 75 (22:6 n-3) a Cutoff value for fatty acid analyses is 40 arbitrary units
  • 5 Clinical Chemistry 53, No. 1, 2007 Fig. 3. A diagnostic algorithm illustrating a suggested evaluation of patients with CF, including fatty acid analysis as a complementary and/or alternative test to the sweat chloride test in patients with CF (28 ). *, Sweat test results must be 80 to be abnormal in infants. **, Sweat test results must be 40 to be normal in infants. respectively. The rationale for this approach is analogous were very similar. The control group for the blinded trial to the protocol for the HIV ELISA screening test, which is showed a lower DHA concentration than the control followed up by a Western blot test for confirmation, with group for the nonblinded trial (Fig. 2), the major differ- the goal of 100% sensitivity in the screening study. ence between the 2 control groups. This finding may be To determine whether plasma fatty acid analysis can attributable to lower fish consumption or fish oil supple- help in the evaluation of CF patients, we compared the mentation in the blinded trial than the nonblinded trial sensitivity, specificity, positive predictive value, and neg- control groups, both of which were randomly selected. ative predictive value of the sweat chloride test reported Because total fatty acid values were very similar in both in 2 different published studies (24, 25 ) with the most CF populations, a value 40 could be used as a clinical favorable diagnostic fatty acid marker in our study cutoff for CF; therefore, patients with a value 40 should (Table 3). The technical failure rate of plasma fatty acid undergo follow-up genetic studies. In the blinded and analysis as a diagnostic test for CF is negligible because nonblinded trials, use of a cutoff of 40 would have blood samples are readily collected. resulted in a genetic study for 4 controls and 1 control,
  • 6 Batal et al.: Plasma Fatty Acids and CF patients with borderline sweat test values will be infor- The Gibson–Cooke Sweat Test (GCST) is the standard mative. In addition, further study is necessary to deter- technique used in the diagnosis of CF. Two previous mine the diagnostic accuracy of plasma fatty acid analysis published studies (24, 25 ) compared the results of the in a CF clinical setting. The data in this initial study, GCST technique with results obtained using different however, indicate that fatty acid analysis is a promising assays for sweat chloride collection and measurement. screening test for CF if sweat chloride testing cannot be The 1st study (24 ) used a cutoff value of 70 mmol/L to performed. differentiate between intermediate and abnormal sweat chloride test results rather than the cutoff value of 60 mmol/L used by the Cystic Fibrosis Foundation (26 ). In References the Mastella et al. study (24 ), 3.6% of the samples did not 1. 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