We are grateful to the individuals with BTHS and their families for participating in our research. We thank the Barth Syndrome Foundation US and the Barth Syndrome UK Trust for their support and for helping with the collection of the blood samples at the annual meeting in Bristol. This study was funded by Barth Syndrome Foundation US, Barth Italia Onlus, and Apulia Region.

Blood samples of healthy donors and heart failure patients were collected at the Policlinic Hospital of Bari (Italy), while samples of BTHS patients were obtained by the National Health Service UK BTHS clinic at Bristol Royal Hospital for Children (UK). Written informed consent of healthy donors, patients, and parents (where appropriate) and approvals by the respective ethics committees were obtained.
NOTE: If not used immediately, blood (in K-EDTA gel tube) can be stored at 4 &#176;C for up t...

<ol>
	<li>Barth, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-linked+cardioskeletal+myopathy+and+neutropenia+(Barth+syndrome):+respiratory-chain+abnormalities+in+cultured+fibroblasts.">X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts.</a> Journal of Inherited Metabolic Disease. 19 (2), 157-160 (1996).</li><li>Steward, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=syndrome+(X+linked+cardiac+and+skeletal+myopathy,+neutropenia,+and+organic+aciduria):+rarely+recognised,+frequently+fatal+[abstract].">syndrome (X linked cardiac and skeletal myopathy, neutropenia, and organic aciduria): rarely recognised, frequently fatal [abstract].</a> Archives of Disease in Childhood. 89, 48 (2004).</li><li>Clarke, S. L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barth+syndrome.">Barth syndrome.</a> Orphanet Journal of Rare Diseases. 8, 23 (2013).</li><li>Zegallai, H. M., Hatch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barth+syndrome:+cardiolipin,+cellular+pathophysiology,+management,+and+novel+therapeutic+targets.">Barth syndrome: cardiolipin, cellular pathophysiology, management, and novel therapeutic targets.</a> Molecular and Cellular Biochemistry. 476 (3), 1605-1629 (2021).</li><li>Taylor, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+presentation+and+natural+history+of+Barth+Syndrome:+An+overview.">Clinical presentation and natural history of Barth Syndrome: An overview.</a> Journal of Inherited Metabolic Disease. 45 (1), 7-16 (2022).</li><li>Miller, P. C., Ren, M., Schlame, M., Toth, M. J., Phoon, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+analysis+to+determine+the+prevalence+of+Barth+syndrome+in+the+pediatric+population.">Bayesian analysis to determine the prevalence of Barth syndrome in the pediatric population.</a> The Journal of Pediatrics. 217, 139-144 (2020).</li><li>Bione, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+X-linked+gene,+G4.5.+is+responsible+for+Barth+syndrome.">A novel X-linked gene, G4.5. is responsible for Barth syndrome.</a> Nature Genetics. 12 (4), 385-389 (1996).</li><li>Whited, K., Baile, M. G., Currier, P., Claypool, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+functional+classes+of+Barth+Syndrome+mutation.">Seven functional classes of Barth Syndrome mutation.</a> Human Molecular Genetics. 22 (3), 483-492 (2013).</li><li>Schlame, M., Ren, M., Xu, Y., Greenberg, M. L., Haller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+symmetry+in+mitochondrial+cardiolipins.">Molecular symmetry in mitochondrial cardiolipins.</a> Chemistry and Physics of Lipids. 138 (1-2), 38-49 (2005).</li><li>Schlame, M., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+Tafazzin,+a+mitochondrial+phospholipid-lysophospholipid+acyltransferase.">The function of Tafazzin, a mitochondrial phospholipid-lysophospholipid acyltransferase.</a> Journal of Molecular Biology. 432 (18), 5043-5051 (2020).</li><li>Schlame, M., Rua, D., Greenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biosynthesis+and+functional+role+of+cardiolipin.">The biosynthesis and functional role of cardiolipin.</a> Progress in Lipid Research. 39 (3), 257-288 (2000).</li><li>Mileykovskaya, E., Dowhan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+membrane+domains+in+prokaryotes+and+eukaryotes.">Cardiolipin membrane domains in prokaryotes and eukaryotes.</a> Biochimica et Biophysica Acta. 1788 (10), 2084-2091 (2009).</li><li>Claypool, S. M., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complexity+of+cardiolipin+in+health+and+disease.">The complexity of cardiolipin in health and disease.</a> Trends in Biochemical Sciences. 37 (1), 32-41 (2011).</li><li>Ren, M., Phoon, C. K., Schlame, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+function+of+mitochondrial+cardiolipin.">Metabolism and function of mitochondrial cardiolipin.</a> Progress in Lipid Research. 55, 1-16 (2014).</li><li>Paradies, G., Paradies, V., Ruggiero, F. M., Petrosillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cardiolipin+in+mitochondrial+function+and+dynamics+in+health+and+disease:+Molecular+and+pharmacological+aspects.">Role of cardiolipin in mitochondrial function and dynamics in health and disease: Molecular and pharmacological aspects.</a> Cells. 8 (7), 728 (2019).</li><li>Acoba, M. G., Senoo, N., Claypool, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+ebb+and+flow+makes+mitochondria+go.">Phospholipid ebb and flow makes mitochondria go.</a> The Journal of Cell Biology. 219 (8), 03131 (2020).</li><li>Schlame, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+abnormalities+in+children+with+Barth+syndrome.">Phospholipid abnormalities in children with Barth syndrome.</a> Journal of the American College of Cardiology. 42 (11), 1994-1999 (2003).</li><li>Wortmann, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+errors+of+metabolism+with+3-methylglutaconic+aciduria+as+discriminative+feature:+proper+classification+and+nomenclature.">Inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature: proper classification and nomenclature.</a> Journal of Inherited Metabolic Disease. 36 (6), 923-928 (2013).</li><li>Ikon, N., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+3-methylglutaconic+acid+in+disorders+of+mitochondrial+energy+metabolism.">On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism.</a> Journal of Inherited Metabolic Disease. 39 (5), 749-756 (2016).</li><li>Kulik, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bloodspot+assay+using+HPLC-tandem+mass+spectrometry+for+detection+of+Barth+syndrome.">Bloodspot assay using HPLC-tandem mass spectrometry for detection of Barth syndrome.</a> Clinical Chemistry. 54 (2), 371-378 (2008).</li><li>Vaz, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+functional+assay+in+blood+spot+to+diagnose+Barth+syndrome+using+the+monolysocardiolipin/cardiolipin+ratio.">An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio.</a> Journal of Inherited Metabolic Disease. 45 (1), 29-37 (2022).</li><li>Bowron, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Barth+syndrome+using+a+novel+LC-MS/MS+method+for+leukocyte+cardiolipin+analysis.">Diagnosis of Barth syndrome using a novel LC-MS/MS method for leukocyte cardiolipin analysis.</a> Journal of Inherited Metabolic Disease. 36 (5), 741-746 (2013).</li><li>Sun, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+assisted+laser+desorption/ionization+time-of-flight+mass+spectrometric+analysis+of+cellular+glycerophospholipids+enabled+by+multiplexed+solvent+dependent+analyte-matrix+interactions.">Matrix assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions.</a> Analytical Chemistry. 80 (19), 7576-7585 (2008).</li><li>Leopold, J., Popkova, Y., Engel, K. M., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+of+useful+MALDI+matrices+for+the+mass+spectrometric+characterization+of+lipids.">Recent developments of useful MALDI matrices for the mass spectrometric characterization of lipids.</a> Biomolecules. 8 (4), 173 (2018).</li><li>Angelini, R., Babudri, F., Lobasso, S., Corcelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF/MS+analysis+of+archaebacterial+lipids+in+lyophilized+membranes+dry-mixed+with+9-aminoacridine.">MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine.</a> The Journal of Lipid Research. 51 (9), 2818-2825 (2010).</li><li>Angelini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomics+of+intact+mitochondria+by+MALDI-TOF+MS.">Lipidomics of intact mitochondria by MALDI-TOF MS.</a> The Journal of Lipid Research. 53 (7), 1417-1425 (2012).</li><li>Angelini, R., Vormieter, G., Corcelli, A., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+method+for+the+determination+of+PC/LPC+ratio+in+intact+horse+serum+by+MALDI-TOF-MS:+an+easy-to-follow+lipid+biomarker+of+inflammation.">A fast method for the determination of PC/LPC ratio in intact horse serum by MALDI-TOF-MS: an easy-to-follow lipid biomarker of inflammation.</a> Chemistry and Physics of Lipids. 183, 169-175 (2014).</li><li>Angelini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+fingerprinting+of+leukocytes+by+MALDI-TOF/MS+as+a+screening+tool+for+Barth+syndrome.">Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome.</a> The Journal of Lipid Research. 56 (9), 1787-1794 (2015).</li><li>Lobasso, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipidomic+approach+to+identify+potential+biomarkers+in+exosomes+from+melanoma+cells+with+different+metastatic+potential.">A lipidomic approach to identify potential biomarkers in exosomes from melanoma cells with different metastatic potential.</a> Frontiers in Physiology. 12, 748895 (2021).</li><li>Angelini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+cholesterol+in+the+brain+by+on-tissue+derivatization+and+quantitative+mass+spectrometry+imaging.">Visualizing cholesterol in the brain by on-tissue derivatization and quantitative mass spectrometry imaging.</a> Analytical Chemistry. 93 (11), 4932-4949 (2021).</li><li>Greco, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+MALDI-TOF+mass+spectrometry+in+clinical+proteomics.">Applications of MALDI-TOF mass spectrometry in clinical proteomics.</a> Expert Review of Proteomics. 15 (8), 683-696 (2018).</li><li>Duncan, M., DeMarco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS:+Emerging+roles+in+pathology+and+laboratory+medicine.">MALDI-MS: Emerging roles in pathology and laboratory medicine.</a> Clinical Mass Spectrometry (Del Mar, Calif). 13, 1-4 (2019).</li></ol>

Barth syndrome is an inborn error of metabolism and a life-changing condition that is likely to be under-diagnosed2,6. As mentioned before, a contributing factor may be the lack of a straightforward diagnostic test. Here, a simple and fast method to measure MLCL/CL ratio by MALDI-TOF/MS in leukocytes for BTHS screening was described. Moreover, MALDI-TOF mass spectrometers are widely distributed among clinical laboratories worldwide and do not require high an...

Barth syndrome (BTHS) is a rare X-linked disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delay, neutropenia, variable mitochondrial respiratory chain dysfunction, and abnormal mitochondrial structure1,2,3,4,5. BTHS has a prevalence of one case per million males with currently less than 250 known cases worldwide, though it is widely accepted that the disease is underdiagnosed2,6. BTHS results from loss-of-function mutations of the Tafazzin (TAFAZZIN) gene localized to chromosome Xq28.127,8 causing deficient remodeling of the mitochondrial phospholipid cardiolipin (CL), a process that normally leads to a highly symmetric and unsaturated acyl composition9,10. CL has been considered the signature lipid of mitochondria, where it is an important constituent of the inner membrane, vital for oxidative phosphorylation (i.e., mitochondrial energy metabolism), supercomplex formation, protein import, and involved in mitochondrial dynamics, mitophagy, and apoptosis11,12,13,14,15,16. Upon TAFAZZIN loss-of-function, CL remodeling fails and specific phospholipid abnormalities arise in mitochondria of BTHS patients: mature CL level (CLm) is decreased, while increased levels of monolysocardiolipin (MLCL) and altered CL acyl composition (i.e., immature CL species, CLi) occur. This brings to a dramatic increase of the MLCL/CL ratio17.
Diagnosis of BTHS is often difficult, as the disorder presents extremely variable clinical and biochemical features and may differ between affected individuals from the same family and within a patient over time3,5. Many BTHS boys show a very high level of urinary excretion of 3-methylglutaconic acid (3-MGCA), but the urine level may be normal or only mildly increased in patients over time3. However, increased 3-MGCA is a feature of various other mitochondrial and non-mitochondrial disorders, such as 3-methylglutaconyl-CoA hydratase deficiency (AUH defect), 3-methylglutaconic aciduria, dystonia-deafness, encephalopathy, Leigh-like (MEGDEL) syndrome, Costeff syndrome, and dilated cardiomyopathy with ataxia (DCMA) syndrome18,19. Hence, the poor specificity of 3-MCGA as a marker for BTHS and the enormous variability in patients render the biochemical diagnosis ambiguous.
Moreover, over 120 different TAFAZZIN mutations have been described causing the disorder5 and, therefore, a genetic diagnosis can be complicated, slow, and expensive. Moreover, molecular analysis of the TAFAZZIN gene can lead to false-negative results in the presence of mutations in noncoding or regulating sequences3. BTHS can be unambiguously tested by determining the relative amounts and distribution of (monolyso-)CL species and confirmed by TAFAZZIN gene sequencing or vice versa.
A practical test for diagnosis is the measurement of the MLCL/CL ratio by High-Performance Liquid Chromatography (HPLC) and Electro Spray Ionization / Mass spectrometry (ESI/MS) analysis in blood spot20,21. Measuring CL level alone is not adequate for diagnosis as some patients have near-normal levels of CL but altered MLCL/CL ratio. Therefore, measurement of MLCL/CL ratio has 100% sensitivity and specificity for BTHS diagnosis21. Another validated method based on HPLC and ESI/MS analysis has been set up on leukocytes22, but the complex chromatographic techniques for separation of lipids previously extracted and the expensiveness of the instruments restrict this analysis to a few clinical laboratories. All these factors, together with the lack of a straightforward diagnostic test, have contributed to the under-diagnosis of the condition.
MALDI-TOF/MS is a further valid tool in lipid analysis23,24.This analytical technique can be used to directly obtain lipid profiles of various biological samples, thus skipping extraction and separation steps25,26,27,28,29, including in tissue sections for MS Imaging applications30. Given this advantage, a simple and fast method to diagnose BTHS by profiling mitochondrial CL in intact leukocytes with MALDI-TOF/MS was developed28. Leukocyte isolation from only 1 mL of whole blood by erythrocyte sedimentation and lysis is straightforward and does not require special equipment or reagents. Furthermore, a fast lipid &#34;mini-extraction&#34; protocol applicable to minute amounts of leukocytes was described to warrant the successful acquisition of spectra having cleaner MS signals with a higher signal-to-noise ratio (S/N) than in those obtained from intact leukocytes28. This further step takes little time and allows for analyses to be reproducible even when carried out on MS instruments with poor sensitivity. In summary, the analytical method described here requires minimal sample preparation because time-consuming and labor-intensive chromatographic lipid separation can be skipped, thereby speeding up the test.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,1&#8242;,2,2&#8242;-tetratetradecanoyl cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>750332</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,1&#8242;2,2&#8242;-tetra- (9Z-octadecenoyl) cardiolipin</td>
			<td>Avanti Polar Lipids</td>
			<td>710335</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-di- (9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>878130</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-sn-glycero-3-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>830845</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-snglycero-3-phospho-(1&#8242;-rac-glycerol)</td>
			<td>Avanti Polar Lipids</td>
			<td>840445</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>1,2-ditetradecanoyl-sn-glycero-3-phospho-L-serine</td>
			<td>Avanti Polar Lipids</td>
			<td>840033</td>
			<td>Lipid standard for MALDI-TOF calibration</td>
		</tr>
		<tr>
			<td>2-Propanol, ACS reagent, &#8805;99.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>190764</td>
			<td></td>
		</tr>
		<tr>
			<td>9-Aminoacridine hemihydrate, 98%</td>
			<td>Acros Organics</td>
			<td>134410010</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, ACS reagent, &#8805;99.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>360457</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform, ACS reagent, &#8805;99.8%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>319988</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran from Leuconostoc spp. Mr 450,000-650,000</td>
			<td>Merck Life Science S.r.l.</td>
			<td>31392</td>
			<td></td>
		</tr>
		<tr>
			<td>Flex Analysis 3.3</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>MALDI-TOF mass spectrometer Microflex LRF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Office</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>OmniPur 10X PBS Liquid Concentrate</td>
			<td>Merck Life Science S.r.l.</td>
			<td>6505-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, ACS reagent, 99.0-100.5%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, ACS reagent, &#8805;99.0%</td>
			<td>Merck Life Science S.r.l.</td>
			<td>S9888</td>
			<td></td>
		</tr>
	</tbody>
</table>

fingerprinting cardiolipin in leukocytes by mass spectrometry for a rapid diagnosis of barth syndrome

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial role in the functioning of the inner membrane. Its metabolite monolysocardiolipin (MLCL) is physiologically nearly absent in the lipid extract of animal cells and its appearance is the hallmark of the Barth syndrome (BTHS), a rare and often misdiagnosed genetic disease that causes severe cardiomyopathy in infancy. The method described here generates a "cardiolipin fingerprint" and allows a simple assay of the relative levels of CL and MLCL species in cellular lipid profiles. In the case of leukocytes, only 1 mL of blood is required to measure the MLCL/CL ratio via matrix-assisted laser desorption ionization - time-of-flight/mass spectrometry (MALDI-TOF/MS) just within 2 h from blood withdrawal. The assay is straightforward and can be easily integrated into the routine work of a clinical biochemistry laboratory to screen for BTHS. The test shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

In this study, a simple and rapid method for isolating leukocytes from 1 mL of whole blood and obtaining CL fingerprinting by MALDI-TOF/MS has been described (see Figure 2). Figure 3 shows the comparison of representative CL fingerprinting of leukocytes, obtained from control subjects and BTHS young boys, in the CL and MLCL mass (m/z) range. Table 1 lists CL and MLCL species detected in these mass spectra.
De...

Watch this Scientific Journal Video about Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome at JoVE.com

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial ...

This method generates a cardiolipin finger printing of leukocytes by MALDI-TOF mass spectrometry and allows the measurement of the ratio between monolysocardiolipin and cardiolipin for a rapid diagnosis of Barth syndrome. The main advantage of this technique is the detection of diagnostic lipid species by a single round of mass spectrometry immediately after the isolation of leukocytes from one milliliter of blood. These and the eye sensitivity and specificity make this assay a suitable diagnostic test to be integrated into the routine work of clinical laboratories to screen for Barth syndrome.Begin by placing blood samples on an orbital shaker for 10 minutes. Next, to 0.9 milliliters of whole blood in a 1.5 milliliter tube, add 0.1 milliliters of 20%dextran. Now pipette and disperse the suspension gently 20 times while avoiding air bubbles and let RBCs sediment for about one hour at room temperature.Using a syringe, collect and transfer the yellow supernatant to a 15 milliliter tube and then centrifuge at 400 times G for 15 minutes at room temperature. After discarding the supernatant, resuspend the pellet containing mainly leukocytes and residual RBCs in 0.6 milliliters of ice cold double distilled water. After approximately 15 seconds, add 0.2 milliliters of 0.6 molar potassium chloride to the cell suspension to restore the correct osmolarity.The short osmotic shock will lyse RBC and leave leukocytes intact. Then adjust the final volume to 2.5 milliliters with single strength PBS. Next, centrifuge the suspension at 400 times G for 15 minutes at room temperature.This is followed by discarding the supernatant and rewashing the leukocyte pellet with 2.5 milliliters of single strength PBS. Repeat centrifuge and discard the supernatant as described previously and resuspend the pellet containing leukocytes in 200 microliters of sterilized double distilled water. Freeze the suspension at minus 80 degrees Celsius or directly perform lipid extraction.Start the extraction by transferring 20 microliters of leukocytes suspension to a 1.5 milliliter tube and spin at 16, 000 times G for 30 seconds. After discarding the supernatant, add 10 microliters of chloroform to the remaining pellet and repeatedly pipette to promote lipid extraction. Now, add 10 microliters of 9-aminoacridine matrix solution to the pellet in chloroform.Pipette and disperse repeatedly to mix. Spin the solution containing lipids and chloroform and 9-aminoacridine matrix solution at 16, 000 times G for 30 seconds. Finally, deposit the supernatant as droplets of 0.35 microliters on the MALDI target to be analyzed and let the droplets air dry at least for 10 to 15 minutes.For lipid analysis, acquire mass spectra of samples in triplicates on a MALDI-TOF mass spectrometer. After calibration with lipid standards, set the analyses in the negative ion mode and optimize the detection mass-to-charge ratio range from 200 thomson to 2, 000 thomson for small molecule analysis. Keep the laser fluence 5%above the threshold to have a good signal-to-noise ratio.For each mass spectrum, apply an average of 2, 000 single laser shots. Acquire spectrum in reflector mode using delayed pulsed extraction. Apply gated matrix suspension to 400 thomson to prevent detector saturation.The figure shows that the defects in the TAFAZZIN gene typically determine a lipid profile specific to Barth syndrome indicated by the appearance of monolysocardiolipin and immature cardiolipin forms together with the reduction of mature cardiolipin forms in the cardiolipin fingerprint. Further, the figure also shows that MALDI-TOF/MS analyses of leukocytes of control subjects typically exhibit only two species of mature cardiolipin. The figure shows that monolysocardiolipin plus immature cardiolipin on mature cardiolipin ratio for Barth syndrome patients was higher than for healthy subjects and heart failure patients.Here, the control groups and Barth syndrome patients are separated by more than one order of magnitude, showing that this method has a strong diagnostic ability for Barth syndrome. To ensure a good assay result, it is crucial to carefully follow all the protocol steps from the leukocytes isolation to the monolysocardiolipin-to-cardiolipin ratio calculation. MALDI-TOF mass spectrometry is useful for obtaining lipid profiles of small amounts of various biological samples to identify lipid biomarkers or pathological alterations.

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Research

JoVE Journal

Biochemistry

2.2K vistas.  Swansea University. Este método genera una huella dactilar de cardiolipina de leucocitos mediante espectrometría de masas MALDI-TOF y permite la medición de la relación entre monolisocardiolipina y cardiolipina para un diagnóstico rápido del síndrome de Barth. La principal ventaja de esta técnica es la detección de especies lipídicas diagnósticas mediante una única ronda de espectrometría de masas inmediatamente después del aislamiento de leucocitos de un mililitro de sangre. Estos y la sensibilida...