Key points are not available for this paper at this time.
The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients. The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, BTHS) (MIM 302060) is an X-linked recessive disorder characterized by infantile or childhood onset of dilated cardiomyopathy, neutropenia, skeletal myopathy (1Barth P. G. Wanders R. J. Vreken P. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome) -MIM 302060. J. Pediatr. 1999; 135: 273-276Google Scholar), abnormal mitochondrial ultrastructure (2Barth P. G. Scholte H. R. Berden J. A. Klei-Van Moorsel J. M. Luyt-Houwen I. E. ‘t Veer-Korthof E. T. Van der Harten J. J. Sobotka-Plojhar M. A. An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes. J Neurol Sci. 1983; 62: 327-355Google Scholar, 3Neustein H. B. Lurie P. R. Dahms B. Takahashi M. An X-linked recessive cardiomyopathy with abnormal mitochondria. Pediatrics. 1979; 64: 24-29Crossref Google Scholar), and variable mitochondrial respiratory chain dysfunction not related to a single respiratory chain complex in skeletal muscle (1Barth P. G. Wanders R. J. Vreken P. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome) -MIM 302060. J. Pediatr. 1999; 135: 273-276Google Scholar, 4Christodoulou J. McInnes R. R. Jay V. Wilson G. Becker L. E. Lehotay D. C. Platt B. A. Bridge P. J. Robinson B. H. Clarke J. T. Barth syndrome: clinical observations and genetic linkage studies. Am J Med Genet. 1994; 50: 255-264Google Scholar) and in cultured fibroblasts (5Barth P. G. Van den Bogert C. Bolhuis P. A. Scholte H. R. H. van Gennip A. Schutgens R. B. Ketel A. G. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts. J. Inherit. Metab. Dis. 1996; 19: 157-160Google Scholar). Biochemical findings include increased urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid, and 2-ethylhydracrylic acid (6Gibson K. M. Sherwood W. G. Hoffman G. F. Stumpf D. A. Dianzani I. Schutgens R. B. Barth P. G. Weismann U. Bachmann C. Schrynemackers-Pitance P. Phenotypic heterogeneity in the syndromes of 3-methylglutaconic aciduria. J. Pediatr. 1991; 118: 885-890Google Scholar, 7Kelley R. I. Cheatham J. P. Clark B. J. Nigro M. A. Powell B. R. Sherwood G. W. Sladky J. T. Swisher W. P. X-linked dilated cardiomyopathy with neutropenia, growth retardation, and 3-methylglutaconic aciduria. J. Pediatr. 1991; 119: 738-747Google Scholar), and moderately decreased serum cholesterol levels. The gene mutated in this disorder is the G4. 5 gene or Tafazzin (TAZ) gene (8Bione S. D’Adamo P. Maestrini E. Gedeon A. K. Bolhuis P. A. Toniolo D. A. A Novel X-linked gene, G4. 5. is responsible for Barth syndrome. Nat. Genet. 1996; 12: 385-389Google Scholar), which is localized on Xq28 (9Bolhuis P. A. Hensels G. W. Hulsebos T. J. M. Baas F. Barth P. G. Mapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28. Am. J. Hum. Genet. 1991; 48: 481-485Google Scholar). In 1997, Neuwald (10Neuwald A. F. Barth syndrome may be due to an acyltransferase deficiency. Curr. Biol. 1997; 7: R465-R466Google Scholar) reported that the TAZ gene shares homology with acyltransferases involved in phospholipid biosynthesis and/or remodeling, suggesting the possibility of abnormalities in glycerophospholipid formation in BTHS. This prompted us to study phospholipid metabolism in cultured skin fibroblasts from BTHS patients. We found reduced levels of cardiolipin (CL) and a disturbance in the remodeling of phosphatidylglycerol (PG) and CL (11Vreken P. Valianpour F. Nijtmans L. G. Grivell L. A. Plecko B. Wanders R. J. Barth P. G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome. Biochem. Biophys. Res. Commun. 2000; 279: 378-382Google Scholar). In particular, the incorporation of linoleic acid into PG and CL was dramatically reduced, whereas the incorporation of other fatty acids into these phospholipids was normal. We also showed that the incorporation of linoleic acid into PG and CL in fibroblasts of patients with a variety of mitochondrial disorders different from BTHS was entirely normal. The biosynthesis of CL in BTHS patients was normal, while its pool size was decreased. The biosynthesis of PG, the precursor in de novo synthesis of CL, and its pool size were both slightly increased in BTHS patients. These results lead to the conclusion that the decreased levels of CL in BTHS patients were due to defective remodeling of CL. In mammals, the biosynthesis of cardiolipin occurs via the cytidinediphosphate-diacylglycerol (CDP-DG) pathway (12Bleyl S. B. Mumford B. R. Thompson V. Carey J. C. Pysher T. J. Chin T. K. Ward K. Neonatal, lethal noncompaction of the left ventricular myocardium is allelic with Barth Syndrome. Am. J. Hum. Genet. 1997; 61: 868-872Google Scholar). Newly formed CL undergoes extensive remodeling by deacylation and reacylation in order to produce the specific C18: 2-C18: 2 and C18: 1-C18: 2 diacyl combinations that are observed in mammalian CL (12Bleyl S. B. Mumford B. R. Thompson V. Carey J. C. Pysher T. J. Chin T. K. Ward K. Neonatal, lethal noncompaction of the left ventricular myocardium is allelic with Barth Syndrome. Am. J. Hum. Genet. 1997; 61: 868-872Google Scholar, 13Schlame M. Rua D. Greenberg M. L. The biosynthesis and functional role of cardiolipin. Prog. Lipid Res. 2000; 39: 257-288Google Scholar, 14Ma B. J. Taylor W. A. Dolinsky V. W. Hatch G. M. Acylation of monolysocardiolipin in rat heart. J. Lipid Res. 1999; 40: 1837-1845Google Scholar, 15Hatch G. M. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells (Review). Int. J. Mol. Med. 1998; 1: 33-41Google Scholar, 16Schlame M. Brody S. Hostetler K. Y. Mitochondrial cardiolipin in diverse eukaryotes. Comparison of biosynthetic reactions and molecular acyl species. Eur. J. Biochem. 1993; 212: 727-735Google Scholar). The aim of the present study was to investigate whether addition of linoleic acid to the growth medium could restore the levels in CL in BTHS fibroblasts. For the analysis of CL and PG molecular species in this study, we used online normal-phase high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) (17Valianpour F. Wanders R. J. A. Barth P. G. Overmars H. van Gennip A. H. Quantitative and Compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients. Clin. Chem. 2002; 48: 1390-1397Google Scholar). Fibroblast cell lines from patients with BTHS and healthy controls were included in this study. Individual patient clinical data are presented in Table 1. TABLE 1Clinical, biochemical and genetic findingsPatientAge at Onset and Presenting SymptomAge in Years at Time of StudyDilated CardiomyopathyMild Proximal Muscle WeaknessNeutropeniaStatural Growth, SDOrganic AciduriaTAZ Gene Mutation11. 3 years11. 2+++−33-mgca, 3-mgra, 2-ehaC441G prematuremuscle weakness20. 5 yearsdied 1. 1+++−23-mgca, 3-mgra, 2-ehaG527 ins Cfailure to thrive31. 1 years14. 5+++−33-mgca, 3-mgra, 2-eha428del13cardiac decompensation46 weeks10. 2+++−23-mgcaG877Tcardiac decompensation51st month nonspecific illness, abnormal cardiac echogram1. 7+ ventricular noncompaction++−23-mgca 3-mgraexon 2, AG →AC splice acceptor site3-mgca, 3-methyl-glutaconic acid; 3-mgra, 3-methylglutaric acid; 2-eha, 2 ethyl-hydracrylic acid; TAZ, Tafazzin. Reference sequence GenBank number NM₀00116. Open table in a new tab 3-mgca, 3-methyl-glutaconic acid; 3-mgra, 3-methylglutaric acid; 2-eha, 2 ethyl-hydracrylic acid; TAZ, Tafazzin. Reference sequence GenBank number NM₀00116. All solutions were of analytical grade and were purchased from Merck (Darmstadt, Germany). (C14: 0) 4-CL (internal standard, IS) was purchased from Avanti lipids (Alabaster, AL). (C18: 2) 4-CL was purchased from Fluka BioChemika (Fluka, Buchs, Switzerland). Linoleic acid (C18: 2) was purchased from Sigma (Sigma, St. Louis, MO). Analytical HPLC LiChrospher Si 60 column (2. 1 × 250 mm, 5 μm particle size) was purchased from Merck. Fibroblasts were grown at 37°C under standard conditions in HAM-F10 medium supplemented with 10% FCS. This medium contains a final concentration of 5–6 μM of linoleic acid (standard growth medium). For dose-dependency experiments, the standard growth medium was supplemented with linoleic acid ranging from 3. 5–50 μM. For the time-dependency experiments, fibroblasts were grown for a time period up to 5 days using the standard growth medium in the presence or absence of 50 μM of additional linoleic acid. Growth media were refreshed every 48 h. Cells were collected after trypsinization, centrifuged at 500 g for 5 min, washed twice with 2 ml of PBS, and stored at –80°C until analysis. The compositional and quantitative analysis of CL and compositional analysis of PG were performed using the method described by Valianpour et al. (17Valianpour F. Wanders R. J. A. Barth P. G. Overmars H. van Gennip A. H. Quantitative and Compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients. Clin. Chem. 2002; 48: 1390-1397Google Scholar). In order to investigate whether the differences in the sample matrix could affect the results, the extraction efficiency, recovery of the method, and ion suppression were determined for fibroblasts. The results of the experiments in BTHS patients and controls were compared by a Student's t-test for all individual analysis. The method used here was originally developed and validated for the quantitative and compositional analysis of CL in platelets (17Valianpour F. Wanders R. J. A. Barth P. G. Overmars H. van Gennip A. H. Quantitative and Compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients. Clin. Chem. 2002; 48: 1390-1397Google Scholar). In order to establish whether the method can also be used successfully in fibroblasts, we determined the extraction efficiency, recovery of the method, and ion suppression in fibroblast samples to verify whether the analysis was matrix dependent. The extraction efficiency of (C18: 2) 4-CL was 78. 1 ± 2. 4% (n = 10). The recovery after HPLC was 92. 8% ± 3. 8 (n = 5). The total recovery of the method was 75. 6% ± 3. 1. Both (C18: 2) 4-CL (analyte) and (C14: 0) 4-CL (IS) ions were suppressed by 23. 1 ± 2. 9% and 25. 9 ± 2. 1% (n = 5), respectively, indicating that (C14: 0) 4-CL can be used for the quantitative analysis of CL in fibroblasts. Control fibroblasts and fibroblasts from a BTHS patient were grown in the presence or absence of additional linoleic acid (50 μM). Mass spectra revealed markedly reduced levels of the different CL molecular species in fibroblasts of BTHS patients compared with controls when the cells were grown in standard medium (Fig. 1B, C). In the presence of 50 μM of linoleic acid, however, the levels of the most abundant CL species were markedly increased in fibroblasts from the BTHS patient (Fig. 1A). The fatty acid composition of the most abundant CL molecular species at m/z 723. 7, 724. 6, and 725. 7 was established by MS/MS (Fig. 1D–1F). The CL molecular species at m/z 723. 7 exclusively contains C18: 2 (Fig. 1D), while the CL species at m/z 724. 6 and 725. 7 contain both C18: 2 as well as C18: 1 (Fig. 1E, F). The contribution of the isotope peaks, however, is likely the reason that the ratio of C18: 2/C18: 1 in Fig. 1E and F is not, as one would expect, equal to three for (C18: 2) 3/ (C18: 1) 1-2H2−-CL or one for (C18: 2) 2/ (C18: 1) 2-2H2−-CL. The possible fatty acid composition of CL molecular species in fibroblasts of control and BTHS patients grown in linoleic acid enriched medium are summarized in Table 2. As indicated in this table, CL molecular species, except the one with m/z 738. 6, have one or more C18: 2 moieties, indicating that linoleic acid is the predominant fatty acid in the CL fraction. TABLE 2Possible fatty acid composition of various CL molecular species and fatty acid composition of various PG molecular species found in fibroblastsm/zCL Molecular Speciesm/zPG Molecular SpeciesaThe fatty acid composition was established using tandem mass spetrometry (MS/MS) analysis. 710. 6 (C18: 2) 3/ (C16: 1) 1-CL745. 6 (C16: 1/C18: 1) -PG711. 7 (C18: 2) 3/ (C16: 0) 1-CL747. 6 (C16: 0/C18: 1) -PG723. 7aThe fatty acid composition was established using tandem mass spetrometry (MS/MS) analysis. (C18: 2) 4-CL769. 6 (C18: 2) 2-PG724. 6aThe fatty acid composition was established using tandem mass spetrometry (MS/MS) analysis. (C18: 2) 3/C18: 1-CL771. 6 (C18: 2/C18: 1) -PG725. 7aThe fatty acid composition was established using tandem mass spetrometry (MS/MS) analysis. (C18: 2) 2/ (C18: 1) 2-CL773. 6 (C18: 1) 2-PG736. 6 ( (C18: 2) 2/C18: 1/C20: 4) -CL775. 6 (C18: 0/C18: 1) -PG737. 7 (C18: 2/ (C18: 1) 2/C20: 4) -CL795. 7 (C18: 1/C20: 4) -PG738. 6 ( (C18: 1) 3/C20: 4) -CL797. 6 (C18: 1/C20: 3) -PG and (C18: 2/C20: 2) -PG799. 7 (C18: 1/C20: 2) -PGCL, cardiolipin; PG, phosphatidylglycerol. a The fatty acid composition was established using tandem mass spetrometry (MS/MS) analysis. Open table in a new tab CL, cardiolipin; PG, phosphatidylglycerol. In order to study whether the effect of linoleic acid was restricted to CL, we also studied the levels and fatty acid composition of other phospholipids. As shown in Fig. 2, different molecular species of PG, the precursor in the biosynthesis of CL, were detected in fibroblasts of BTHS patients. No differences were observed in the PG molecular species between the control subjects and BTHS patients when fibroblasts were grown in standard or enriched medium (data not shown). In the presence of linoleic acid, (C18: 2) 2-PG (m/z 769. 6) considerably increased compared with the other PG molecular species in both patient and control cell lines (Fig. 2A, B; shown only for the BTHS patient). The fatty acid composition of the observed molecular species of PG was established as shown in Fig. 2C–2F. The mass at m/z 769. 6 contains C18: 2 (linoleic acid) exclusively. The mass at 797. 6 represents two different fatty acid compositions. The most abundant one is C18: 1/C20: 3 as shown in Fig. 2D, where the other composition, C18: 2/C20: 2, is less abundant. The peaks at m/z 747. 6 and m/z 773. 6 correspond to the fatty acid compositions C16: 0/C18: 1 and C18: 1/C18: 1, respectively, as shown in Fig. 2E and F. Compositional analysis of other phospholipid major classes showed that these all contained a higher amount of linoleic acid when cells were grown in the presence of enriched medium (data not shown), indicating that the effect of linoleic acid was not restricted to CL and PG. The results of the linoleic acid supplementation studies are shown in Fig. 3and Table 3. In order to study the dose-dependent effect of linoleic acid on the CL levels in fibroblasts, cells from controls and patients were grown for 3 days in the presence of increasing concentrations of linoleic acid in the growth medium (Fig. 3A). Figure 3A shows a dose-dependent increase in the levels of CL in controls and patients. In the presence of 50 μM of added linoleic acid, the CL levels in the patients approached the normal range of CL in nontreated controls. In order to study the time-dependent effect of linoleic acid on the CL levels in fibroblasts, cells were grown in the absence (Fig. 3B) or presence (Fig. 3C) of 50 μM of added linoleic acid in growth medium during a period of up to 5 days. As shown in Fig. 3B, the levels of CL remained virtually stable when the cells were grown in the absence of added linoleic acid, both in control and the patients’ cell lines, although the levels of CL in patient cells were considerably lower. In contrast, supplementation with linoleic acid resulted in a time-dependent increase of CL levels in both BTHS and control cell lines (Fig. 3C). TABLE 3CL-levels in fibroblasts of control and BTHS patients after 3 days of growth in standard or linoleic acid enriched mediumCL Molecular SpeciesStandard Growth MediumaMean ± SD. Enriched Growth MediumaMean ± SD. , bFifty micromoles of linoleic acid was added to the standard medium. P ValuescP values of standard medium versus enriched medium were obtained using all individual data in Student's t-test. nmol/mg proteinControl (n = 10) Control (n = 5) (C18: 2) 4-CL0. 78 ± 0. 143. 88 ± 0. 410. 0000000000002 (C18: 2) 3 (C18: 1) 1-CL0. 96 ± 0. 322. 32 ± 0. 510. 00001 (C18: 2) 2 (C18: 1) 2-CL1. 74 ± 0. 594. 32 ± 0. 280. 0000001Barth syndrome (n = 5) Barth syndrome (n = 5) (C18: 2) 4-CL0. 09 ± 0. 041. 28 ± 0. 210. 00000002 (C18: 2) 3 (C18: 1) 1-CL0. 18 ± 0. 141. 16 ± 0. 170. 0000002 (C18: 2) 2 (C18: 1) 2-CL0. 22 ± 0. 111. 59 ± 0. 190. 00000001a Mean ± SD. b Fifty micromoles of linoleic acid was added to the standard medium. c P values of standard medium versus enriched medium were obtained using all individual data in Student's t-test. Open table in a new tab To establish whether the effect of linoleic acid on the levels of CL was reversible, cells from controls and patients were first grown in the presence of supplemented linoleic acid for 3 days. Subsequently, the medium was replaced by standard medium and cells were grown for 5 days. the of the supplemented the levels of CL to values in both BTHS and control cell lines, that the linoleic acid effect is (Fig. Table 3 the results of the CL levels ± in fibroblasts of both control and BTHS patient after a in the presence or absence of added linoleic acid. The linoleic acid supplementation resulted in a increase of the three most abundant molecular species in BTHS and control cells CL and PG are for mitochondrial M. B. formation and reacylation in rat J. Scholar, V. Greenberg M. L. in J. 2000; Scholar, M. E. M. of mitochondrial phospholipids an of of of the A genetic for the study of phospholipid in Biol. Chem. Scholar, A. F. G. Hatch G. M. remodeling in a fibroblast cell in 1997; Scholar, M. for in complex and of the mitochondrial respiratory Biol. Chem. Scholar, of cardiolipin to 1993; Scholar) and CL levels are markedly reduced in BTHS patients (11Vreken P. Valianpour F. Nijtmans L. G. Grivell L. A. Plecko B. Wanders R. J. Barth P. G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome. Biochem. Biophys. Res. Commun. 2000; 279: 378-382Google Scholar, F. Wanders R. J. A. Barth P. G. Overmars H. van Gennip A. H. Quantitative and Compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients. Clin. Chem. 2002; 48: 1390-1397Google Scholar), we studied the effect of linoleic acid supplementation on CL levels in cultured skin fibroblasts of BTHS patients and healthy controls. The in this study was whether the levels of CL in BTHS patients could be restored when the fibroblasts were grown in the presence of high of linoleic acid. Comparison of data for the CL analysis in fibroblasts with the CL analysis in platelets (17Valianpour F. Wanders R. J. A. Barth P. G. Overmars H. van Gennip A. H. Quantitative and Compositional study of cardiolipin in platelets by electrospray ionization mass spectrometry: application for the identification of Barth syndrome patients. Clin. Chem. 2002; 48: 1390-1397Google Scholar) shows that this method is for the compositional and quantitative analysis of CL in fibroblasts. As shown in this was a and dose-dependent increase of the CL levels when the cells were grown in the presence of linoleic acid. This to the of the CL levels to values in skin fibroblasts of patients from BTHS. We observed that the linoleic acid of all major PG, which is the precursor in de novo synthesis of CL, increased when cells were grown in the presence of linoleic acid. The levels of (C18: 2) 2-PG increased considerably in indicating that the increased levels of CL from de novo synthesis of CL from This would lead to the formation of CL molecular species with a higher of linoleic acid, which is observed after on this one would that both control and BTHS cells would the CL values linoleic acid In contrast, the CL levels in BTHS fibroblasts after linoleic acid treatment were considerably in controls. This might be by the remodeling of CL G. M. Cardiolipin: biosynthesis, remodeling and trafficking in the heart and mammalian cells (Review). Int. J. Mol. Med. 1998; 1: 33-41Google Scholar). CL remodeling via a deacylation which is by by a reacylation which is by specific is that the TAZ gene are responsible for the the first of CL remodeling is in BTHS this might lead to of formed CL, which be into CL of the This might the differences in CL levels in control versus BTHS cells after supplementation with linoleic acid. The higher levels of CL in both cell lines to the values when the enriched medium was replaced by standard growth indicating that the observed increased levels of CL were by the supplementation of linoleic acid. The results described in this that a treatment on supplementation of linoleic acid may lead to an increase of CL levels in different and that this may be beneficial to BTHS patients. We have a clinical to investigate whether the treatment described for fibroblasts in can also be under in conditions and whether this treatment is beneficial for the To this end, the patients a enriched diet and the CL of platelets was in time that revealed a increase in CL levels. The in effect of the increase in CL be by of the cardiac and The are to P. and P. for cell The the for Barth syndrome cardiolipin standard phosphatidylglycerol Tafazzin
Valianpour et al. (Sat,) studied this question.