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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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-functio....

Protocol

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 °C for up t.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
1,1′,2,2′-tetratetradecanoyl cardiolipinAvanti Polar Lipids750332Lipid standard for MALDI-TOF calibration
1,1′2,2′-tetra- (9Z-octadecenoyl) cardiolipinAvanti Polar Lipids710335Lipid standard for MALDI-TOF calibration
1,2-di- (9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamineAvanti Polar Lipids878130Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-sn-glycero-3-phosphateAvanti Polar Lipids830845Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-snglycero-3-phospho-(1′-rac-glycerol)Avanti Polar Lipids840445Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-sn-glycero-3-phospho-L-serineAvanti Polar Lipids840033Lipid standard for MALDI-TOF calibration
2-Propanol, ACS reagent, ≥99.5%Merck Life Science S.r.l.190764
9-Aminoacridine hemihydrate, 98%Acros Organics134410010
Acetonitrile, ACS reagent, ≥99.5%Merck Life Science S.r.l.360457
Chloroform, ACS reagent, ≥99.8%Merck Life Science S.r.l.319988
Dextran from Leuconostoc spp. Mr 450,000-650,000Merck Life Science S.r.l.31392
Flex Analysis 3.3Bruker DaltonicsSoftware
MALDI-TOF mass spectrometer Microflex LRFBruker Daltonics
Microsoft ExcelMicrosoft OfficeSoftware
OmniPur 10X PBS Liquid ConcentrateMerck Life Science S.r.l.6505-OP
Potassium chloride, ACS reagent, 99.0-100.5%Merck Life Science S.r.l.P3911
Sodium chloride, ACS reagent, ≥99.0%Merck Life Science S.r.l.S9888

References

  1. Barth, P. G., et al. X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts. Journal of Inherited Metabolic Disease. 19 (2), 157-160 (1996).
  2. Steward, C. G., et al.

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