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1 Academic Medical Center, University of Amsterdam, Laboratory Genetic Metabolic Diseases, Emma Children’s Hospital, and the Department of Clinical Chemistry, 1100 DE Amsterdam, The Netherlands.
aAddress correspondence to this author at: Laboratory of Genetic Metabolic Diseases, F0-233, PO Box 22700, 1100 DE Amsterdam, The Netherlands. Fax 31-20-6962596; e-mail a.h.vangennip{at}amc.uva.nl.
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Abstract |
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Methods: We used HPLC and online electrospray ionization mass spectrometry (HPLC-ESI-MS) to quantify CL molecular species. The CL content of platelets was studied in blood samples of BTHS and non-BTHS patients. Control blood samples drawn from healthy adults were collected and analyzed within 24 h (n = 10) and 48 h (n = 10) to characterize any effect of sample shipping time on the CL content in platelets. Samples were collected from children 1–10 years of age who were not affected by BTHS (n = 6) and from BTHS patients (n = 4) and analyzed within 24 h. Results for all four groups were compared by a Student t-test for all individual analyses.
Results: We found different CL molecular species, e.g., (C18:2)4-CL. BTHS patients had a specific decrease of tetralinoleyl-CL concentrations in platelets (0.1–0.5 nmol/mg of protein; n = 4) compared with all control groups (2.3–5.5 nmol/mg of protein; n = 26). Only minor differences were observed among the different control groups.
Conclusions: Quantitative and compositional analyses of CL in platelets by the proposed method allow identification of BTHS patients more rapidly than gene analysis or analysis of CL in cultured skin fibroblasts. The abnormality of CL may explain the abnormal mitochondrial function observed in BTHS. The differences between the control groups did not cause any complication.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The trivial name "cardiolipin" was derived because this phospholipid was first found in animal hearts, where it is especially abundant. However, CL is found in the mitochondria of animal species, yeasts, and bacteria (3). The biosynthetic pathway of CL is similar to those of other phospholipids; it passes through the common intermediates, phosphatidic acid and phosphatidyl-CMP. However, the final step is a unique reaction that is different between prokaryotes and eukaryotes (4). In prokaryotes, diphosphatidylglycerol synthase catalyses the transfer of a phosphatidyl moiety from one phosphatidylglycerol (PG) to the free 3'-hydroxyl group of another PG (3)(4)(5), whereas eukaryotic diphosphatidylglycerol synthase links an activated phosphatidyl moiety (phosphatidyl-CMP) to PG (3)(5)(6)(7). In animal tissues, CL contains almost exclusively 18-carbon fatty acids, and 80% of these are typically linoleic acid [C18:2(n-6)] (7). This also appears to be true for the higher plants (7). Yeast PG differs by having more C16:1 and C18:1 fatty acids (4), whereas the bacterial lipid contains saturated and monoenoic fatty acids with 14–18 carbons (8)(9). The precise function of CL is still a matter of debate.
Variations in the concentrations of CL or its fatty acid composition affect the function of respiratory chain complexes (10). Recently, we showed defective incorporation of linoleic acid into CL in cultured skin fibroblasts of patients suffering from Barth syndrome (BTHS) (10). BTHS (MIM 302060) is an X-linked recessive disorder characterized by infantile or childhood onset of dilated cardiomyopathy, neutropenia, skeletal myopathy (11), abnormal mitochondrial ultrastructure (12)(13), and variable mitochondrial respiratory chain dysfunction. This dysfunction is evidently unrelated to a single respiratory chain complex in skeletal muscle (11)(14) and in cultured fibroblasts (15). Biochemical findings of BTHS include increased urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid, and 2-ethylhydracrylic acid (16)(17) and moderately decreased serum cholesterol concentrations (17). The gene involved in the disease is the G4.5 gene (18), or tafazzin (TAZ) gene (18), which is localized at Xq28 (19). The TAZ gene has 11 exons and two ATG initiation sites (on exons 1 and 3). Diagnosis of BTHS has been by molecular analysis through the identification of mutations in the G4.5/TAZ gene.
A biochemical method for BTHS was lacking until recently, when we presented a biochemical test based on the incorporation of linoleic acid into the phospholipids of cultured skin fibroblasts to distinguish between BTHS patients and controls (10). However, this method (10) and other conventional analytic methods for quantitative or compositional analysis of CL (20)(21)(22)(23) are typically multistep procedures. Furthermore, they are time-consuming, and the analyses require a large amount of mitochondrial membrane. The availability of a simple, rapid method for the microanalysis of CL is important for investigation of CL metabolism in various tissues. Our primary goal was to develop a rapid method to analyze CL isolated from platelets for research and diagnostic purposes. We used platelet samples from BTHS patients to show the usefulness of this method.
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Materials and Methods |
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samples
To check the possible effect of sample shipping time, blood samples were collected (2 mL) with EDTA as an anticoagulant and kept at room temperature for 24 h (24-h blood samples; n = 10) and 48 h (48-h blood samples; n = 10). Blood samples (2 mL) from patients with BTHS were collected and analyzed within 24 h (n = 4; Table 3
). Blood samples (2 mL) from children (age range, 2–10 years; n = 6) without BTHS were collected and analyzed in the same manner as those from BTHS patients. All samples were collected according to institutional guidelines for blood sampling, including procurement of informed consent of the persons involved or their representatives.
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procedures
Isolation of platelets.
EDTA-anticoagulated whole blood was kept at room temperature and mixed continually. The blood (2 mL) was centrifuged at 600g for 10 min at room temperature, and the resultant platelet-rich plasma was then centrifuged at 11 000g (4 °C). The pellets were washed twice with phosphate-buffered saline, centrifuged at 11 000g (4 °C), and subsequently stored at -80 °C.
Extraction of lipids.
Lipids were extracted using a modification of the method of Folch et al. (24). In brief, platelet pellets were suspended in 1 mL of water and sonicated twice for 10 s; 50 µL was then removed and used for protein measurement. We then added 50 µL of a 7.8 µmol/L solution of IS [(C14:0)4-CL] in chloroform–methanol (2:1 by volume) and 3 mL of a chloroform–methanol solution (2:1 by volume) to the remaining cell suspension. The mixture was then shaken vigorously and put on ice for 15 min. Phase separation was achieved by centrifugation at 1000g for 5 min. The organic layer was removed, and the aqueous layer was extracted again with 3 mL of a chloroform–methanol solution (2:1 by volume). The organic layers were combined and washed with 1 mL of chloroform–methanol–water (1:46:47 by volume). The organic layer was evaporated under a stream of nitrogen gas. The residue was dissolved in 150 µL of chloroform–methanol–water (50:45:5 by volume) and designated the lipid extract.
HPLC.
The HPLC system consisted of a HP1100 series binary-gradient pump, a vacuum degasser, a column temperature controller (all from Hewlett-Packard), and a Gilson 231 XL autosampler (Gilson). The column temperature was maintained at 22 °C. The lipid extract was injected onto a 2.1 x 250-mm silica column (5-µm particle diameter; Merck). The phospholipids were separated from interfering compounds by a linear gradient between solution B (chloroform) and solution A (methanol–water; 9:1 by volume). Both solutions contained 0.1 mL of 250 mL/L aqueous ammonia per liter of solution. The gradient (0.3 mL/min) was as follows: 0–3 min, 20% A to 100% A; 3–8 min, 100% A; 8–8.1 min, 100% A to 0% A; and 8.1–14 min, equilibration with 0% A. All gradient steps were linear, and the total analysis time, including equilibration, was 14 min. We used a splitter between the HPLC column and the mass spectrometer, and 30 µL/min of eluant was introduced into the mass spectrometer. An electrically operated valve ensured that the eluant was introduced into the mass spectrometer from 4 to 8 min only.
Mass spectrometry (MS).
A Quattro II triple-quadrupole mass spectrometer (Micromass) was used in the negative electrospray ionization (ESI) mode. Nitrogen was used as the nebulizing gas, and argon was used as the collision gas at a pressure of 0.25 Pa. The capillary voltage was 3 kV, the source temperature was set at 80 °C, and the optimal cone-voltage energy was 30 V. Daughter fragments of m/z 723.6, 724.6, and 725.6 (CL molecular species) were obtained using an optimal collision energy of 40 eV. High cone-voltage energy (100 V) was used to obtain the fatty acid composition in the CL fraction. Both tetralinoleyl-CL (C18:2)4-CL and (C14:0)4-CL (IS) were measured by single-ion monitoring of their double-charged ions in negative mode.
Validation.
The HPLC and mass spectrometer settings were optimized with a 10 µmol/L solution of commercially available (C14:0)4-CL (synthetic) and (C18:2)4-CL in chloroform–methanol–water (50:45:5 by volume) containing 0.1 mL/L of 250 mL/L aqueous ammonia. Of this calibrator solution, 5 µL was injected into the HPLC column, and one-tenth of the eluting solution was introduced into the mass spectrometer. The purity of the commercially available (C18:2)4-CL was assessed by measuring all CL molecular species in the sample and calculating the purity of (C18:2)4-CL as a percentage of the total.
The linear response and detection limit for (C18:2)4-CL were established by injection of calibration mixtures with different concentrations of this analyte and a constant concentration of (C14:0)4-CL as the IS. The amount of (C18:2)4-CL in the samples was calculated as follows: amount of (C18:2)4-CL/mg of protein = (peak area analyte x IS concentration x response factor)/(peak area IS x mg of protein used in the assay). The extraction efficiencies for both the IS and analyte were obtained by comparing their peak intensities in the extracted calibration mixtures with those in the unextracted calibration mixtures with the same final concentrations without extraction.
The intraassay (within-day) variation of the method was determined by measuring 10 times a nonenriched sample and a sample enriched with commercially available (C18:2)4-CL at low (2 nmol/mg of protein), medium (10 nmol/mg of protein), and high (100 nmol/mg of protein) concentrations. The interassay (between-day) variation was determined by measuring blank samples and samples enriched with commercially available (C18:2)4-CL (10 and 100 nmol/mg of protein) over 3 separate weeks. The recovery efficiency of the method was established by measuring 10 different samples before and after enrichment with a known amount of commercially available (C18:2)4-CL. We investigated ion suppression from interfering substances by comparison of the peak intensities of both (C18:2)4-CL and (C14:0)4-CL in the enriched samples with those of calibrator solutions with similar concentrations.
In all experiments, no special precautions (e.g., cleaning of the high-voltage lens and sample cone) were taken to optimize the detection limit of the MS system. Platelets from different control groups and from BTHS patients were analyzed to establish the usefulness of this method. The results of these experiments were compared by a Student t-test for all individual analyses.
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Results |
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shows the CL molecular species isolated from a platelet sample detected by tandem MS in the elution fraction (peak) of CL. The peak at m/z 619.6 belongs to [(C14:0)4-2H]2--CL (IS). The peaks at m/z 723.6, 724.6, and 725.6 belong to [(C18:2)4-2H]2--CL, [(C18:2)3/(C18:1)1-2H]2--CL, and [(C18:2)2/(C18:1)2-2H]2--CL, respectively. Fig. 2
shows the daughter-fragment spectra of these molecular species. The m/z 723.6 peak has C18:2 (m/z 279.2; Fig. 2
) exclusively as a side chain, whereas the peaks at m/z 724.6 and 725.6 have both C18:2 and C18:1 (m/z 281.3; Fig. 2
) as side chains. However, the peak at m/z 724.6 corresponds to the second isotope peak of m/z 723.6, and the peak at m/z 725.6 corresponds to the second isotope peak of m/z 724.6 and to the fourth isotope peak of m/z 723.6. This is probably the reason that the ratio C18:2/C18:1 in Fig. 2
, panels B and C, is not, as one would expect, equal to 3 for [(C18:2)3/(C18:1)1-2H]2--CL (m/z 724.6) or 1 for [(C18:2)2/(C18:1)2-2H]2--CL.
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The peaks 0.5-Da from each peak correspond to the first isotope peaks. The peak clusters at m/z 711.6 and 736.6, shown in more detail in Fig. 3
, also have their first isotope peaks at a distance of 0.5 Da, which indicates that the corresponding ions carry a double charge. These ions may have the structures indicated in Table 1
. Fig. 4
shows high cone-voltage spectra (100 V) of the CL fractions from a control and a patient sample. The CL molecules were fragmented and the fragments detected. The peaks at m/z 253.5, 255.2, 279.2, 281.3, 283.3, and 303.3 belong to the fatty acids palmitoleic (C16:1), palmitic (C16:0), linoleic (C18:2), oleic (C18:1), stearic (C18:0), and arachidonic (C20:4) acid. Linoleic acid was the most abundant fatty acid in the control sample, whereas it was almost absent in the patient sample. The presence of these fatty acids confirms the existence of other CL molecular species that have fatty acids other than C18:2 and C18:1 as side chains, as indicated in Table 1
and Figs. 1
and 2
.
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validation
The response factor for (C18:2)4-CL was determined by using the slope and y-intercept of the calibration curve, which were obtained from a linear least-squares regression for analyte/IS peak-area ratio vs analyte concentration (r2 >0.982). Because of the differences between the IS and the analyte and because detector response is dependent on these differences, we used this response factor to calculate the concentrations of (C18:2)4-CL in our samples. The ratio between the analyte concentration and the protein concentration of the sample was linear up to at least 0.04 g/L protein (r2 >0.966). When the protein concentrations of the samples exceeded 4 g/L, the samples were diluted before extraction.
The detection limits were established and defined as the lowest signals with a signal-to-noise ratio of 3. The detection limits obtained were 0.7 µmol/L for the IS and 0.4 µmol/L for the commercially available (C18:2)4-CL. However, the lowest concentration with a measurable signal-to-noise ratio of 3 was 
10 nmol/g of protein, when a sample with at least 0.3 x 10-3 g/L protein was available.
The extraction efficiency for (C18:2)4-CL was 82.6% ± 3.9% for nonenriched samples, 89.3% ± 2.6% for samples enriched with low concentrations, 96.7% ± 4.2% for samples enriched with medium concentrations, and 94.5% ± 5.4% for samples enriched with high concentrations of (C18:2)4-CL. The extraction efficiency for (C14:0)4-CL (IS) was in the same range. The variation in retention times for both components (analyte and IS) was <0.2%.
The intraassay variation was 6.8% for nonenriched samples, 3.7% for samples enriched with low concentrations, 5.9% for samples enriched with medium concentrations, and 6.1% for samples enriched with high concentrations of (C18:2)4-CL. The interassay CV was 11% for nonenriched samples and 14% for enriched samples. The recovery efficiency of the method was 96.4% ± 2.1%. The (C18:2)4-CL and (C14:0)4-CL ions were suppressed by 33.5% ± 4.1% and 31.9% ± 3.4%, respectively.
application to samples from patients and controls
Using this method, we measured the concentrations of (C18:2)4-CL in platelets prepared from blood samples of healthy, adult volunteers after 24 (n = 10) and 48 (n = 10) h, from a group of children (ages 2–10 years; n = 6), and from a group of BTHS patients (n = 4). Table 2
summarizes the clinical, biochemical, and genetic findings for the BTHS patients. Fig. 5
shows chromatograms of doubly charged (C18:2)4-CL from a control sample and a BTHS patient sample. (C18:2)4-CL is markedly decreased in the patient sample. Table 3
shows the quantification results for (C18:2)4-CL in the various patient and control groups. We noted a significant difference between the BTHS patients and the control groups. We also observed a difference between results for blood samples from the 24-h adult group vs the 48-h adult controls and the pediatric group (P <0.03).
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Discussion |
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The major classes of phospholipids differ in the polar head group, which makes it possible to separate them on a normal-phase silica-based column. However, CL and other phospholipids were separated in a short time from interfering nonpolar components by a binary solvent system, without any complications in mixability of the gradient components. In addition, this method requires only a small amount of biological material. The solvent composition forms a very good matrix for the ionization of CL in ESI-MS. Using this method, we measured CL molecular species in different biological samples, such as platelets, fibroblasts, lymphoblasts, yeast cells, and several other tissues (data not shown). The method was not optimized for the measurement of other phospholipids.
We used platelets in this study because they are easily isolated from whole blood compared with other blood cells or fibroblasts, which must be cultured for several weeks before use. For the quantitative measurement of CL, we determined the detector response factor to correct the measured concentrations of (C18:2)4-CL before each set of analyses. We also defined and measured a pool sample of healthy controls before each set of analyses. Validation data show that our method can be used for the compositional analysis of CL and quantitative analysis of (C18:2)4-CL. Our data for the different CL molecular species in platelets are comparable with CL molecular species found in other human tissues (4). However, because internal calibrators labeled with stable isotopes are lacking, precautions, such measuring the response factor and performing recovery experiments for both the IS and the analyte, are advised for the quantification of CL molecular species by ESI-MS.
In yeast, different mitochondrial dysfunctions occur when CL synthase or phosphatidylglycerophosphate synthase undergo mutation, which indicates that CL plays an important role in mitochondrial function. However, the exact function of CL is still unclear. Our newly developed method can be used to clarify the function of CL in relation to mitochondrial disorders.
Because BTHS, an X-linked recessive disorder with several mitochondrial dysfunctions (12), has been associated with a possible remodeling defect of CL (10), we used our method to establish a procedure for the identification of BTHS patients. Although the diagnosis of BTHS is usually established by molecular studies, a screening method for BTHS is greatly needed because the clinical picture of this syndrome is nonspecific, because gene analysis is time-consuming, and because the number of patients is high. Recently, we introduced a biochemical test that incorporates radiolabeled linoleic acid into CL or the PG pool of fibroblasts (10). Both CL and PG had normal de novo synthesis. A reduced incorporation of linoleic acid into both PG and CL, however, was observed in BTHS patients, whereas incorporation of the other fatty acids, such as C16:0, C18:1, and C20:4 was within the range of values obtained in a healthy population. The total CL pool was reduced, whereas the total PG pool was slightly increased. Because PG can be separated by one-dimensional thin-layer chromatography more easily than CL from other phospholipids and natural lipids, we suggested the incorporation of linoleic acid into the PG pool as a biochemical test. However, both of these methods are time-consuming (days to weeks) and are therefore not very attractive for use as a screening tool to pinpoint patients suspected of BTHS; our new method, in contrast, takes only 5 h for 10 samples. Because the clinical and biochemical abnormalities in BTHS patients are nonspecific, an easy and rapid method for BTHS detection is very attractive. As shown in Table 3
, BTHS patients and controls showed a significant difference in CL values. The differences between individual samples within each control group show that the time between blood collection and analysis can cause minor differences, which could be caused by platelet activation during sampling. We therefore suggest that the platelets be isolated immediately after blood collection. However, differences between control groups did not cause any complications with respect to the identification of patients with BHTS.
Because CL is an exclusive mitochondrial inner-membrane phospholipid involved in various mitochondrial functions (1)(2)(3), markedly decreased CL values can explain the abnormal mitochondrial function observed in BTHS patients. We conclude that the analysis of CL in platelets by HPLC-ESI-tandem MS is a rapid and simple method to identify patients with BTHS. However, no evidence is yet available to confirm that the decreased CL concentration is a primary effect of a mutation in the TAZ gene. Thus, at this time, gene mutation analysis may be needed to complete the diagnosis.
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Acknowledgments |
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Footnotes |
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References |
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