Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome

Abstract

Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card-iolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a "CL fingerprint" and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells.

Keywords: cardiomyopathy; lysophospholipids; mass spectrometry; matrix-assisted laser desorption/ionization; mitochondria; phospholipids; phospholipids/metabolism; tafazzin; time-of-flight.

Fig. 1.

Work flow chart (left) and graphical abstract (right). From the blood withdrawal to the test results, only 2 h are necessary. MALDI-TOF/MS-based BTHS diagnosis in four steps: 1) native sample deposition, 2) matrix layering, 3) laser desorption and ionization, and 4) lipid fingerprint analysis.

Fig. 2.

BTHS diagnostic leukocyte CL fingerprints. Lipid profiles of intact leukocytes of a healthy control (CTRL, upper) and a representative BTHS patient (BTHS, lower) are shown. Peaks of CL and MLCL species considered in this study are indicated (in red). The CLm form is highlighted in turquoise. Peaks labeled with an asterisk are assigned to gangliosides and are not of interest for the present study. Spectra were acquired with the Bruker Microflex RLF mass spectrometer.

Fig. 3.

Remodeling enzymatic pathway of CL. iPLA2γ, calcium-independent phospholipase A2 γ.

Fig. 4.

(MLCL + CLi)/CLm ratios in controls and BTHS patients. Error bars indicate SDs obtained in measurements of three spectra for each subject [24 healthy donors as controls (CTRL) and 8 patients (BTHS)]. Ratios have been calculated from CL fingerprints of either intact leukocytes (intact) or after the addition of organic solvents to quickly solubilize lipids (extract), as described in Materials and Methods. A and B: Results obtained by using the low-performance Bruker Microflex RLF mass spectrometer. C: Results obtained by Bruker Autoflex II, with the same number of patients’ samples but with a reduced number of healthy controls. High-p. MS, high-performance mass spectrometry. The y-axis shows a logarithmic scale. Upper cutoff values of controls, calculated as in Bowron et al. (26), are 4.21 for A, 0.64 for B, and 1.16 for C.

Fig. 5.

Compositional distances of CL fingerprints of controls and BTHS patients. The same samples considered in Fig. 4 have been used to estimate differences in molecular composition of CL and its lysoderivative species. Average values ± SDs are provided for controls (CTRL) and typical BTHS patients (BTHS). A and B: Results obtained by using the low-performance Bruker Microflex RLF mass spectrometer. C: Results obtained by Bruker Autoflex II. High-p. MS, high-performance mass spectrometry.