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

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 structure^1,2,3,4,5. 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 underdiagnosed^2,6. BTHS results from loss-of-function mutations of the Tafazzin (TAFAZZIN) gene localized to chromosome Xq28.12^7,8 causing deficient remodeling of the mitochondrial phospholipid cardiolipin (CL), a process that normally leads to a highly symmetric and unsaturated acyl composition^9,10. 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 apoptosis^{11,12,13,14,15,16}. 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 time^3,5. 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) syndrome^18,19. 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 spot^20,21. 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 analysis^23,24.This analytical technique can be used to directly obtain lipid profiles of various biological samples, thus skipping extraction and separation steps^{25,26,27,28,29}, 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 "mini-extraction" 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.

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 to 24-48 h.

1. Isolation of leukocytes by dextran sedimentation of red blood cells (RBCs)

1. Put blood samples (in K-EDTA) on an orbital shaker for 10 min to mix blood.
2. To 0.9 mL of whole blood in a 1.5 mL tube, add 0.1 mL of 20% dextran solution (molecular mass > 100 kDa, in 0.9% NaCl). Pipette and disperse the suspension gently 20 times while avoiding air bubbles that would otherwise retain RBCs at the top of the tube. Let RBCs sediment for about 1 h at room temperature.
3. Using a syringe, collect and transfer the yellow supernatant to a 15 mL tube, and then centrifuge at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor).
4. Discard the supernatant and resuspend the pellet containing mainly leukocytes and residual RBCs in 0.6 mL of ice-cold bi-distilled water (ddH₂O).
5. After ~15 s, add 0.2 mL of 0.6 M KCl to the cell suspension to restore the correct osmolarity. The short osmotic shock will lyse RBC and leave leukocytes intact. Adjust the final volume to 2.5 mL with 1x PBS.
6. Centrifuge the suspension at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor). Discard the supernatant and wash the leukocyte pellet again with 2.5 mL of 1x PBS.
7. Centrifuge as in step 1.6 and discard the supernatant. Resuspend the pellet containing leukocytes in 200 µL of sterilized ddH₂O.
   NOTE: In the authors' experience, this corresponds to a protein content ranging from 200 µg to 400 µg.
8. Freeze the suspension at -80 °C or directly perform lipid extraction as follows.

2. "Mini-extraction" of lipids from isolated leukocytes

1. Transfer 20 µL of leukocytes suspension (about 20-40 µg proteins) to a 1.5 mL tube and spin at 16,000 x g for 30 s.
2. Discard the supernatant, add 10 µL of CHCl₃ to the remaining pellet, and pipette repeatedly to promote lipid extraction.
3. Finally, add 10 µL of 9-aminoacridine (9-AA) matrix solution (10 mg/mL 9-AA in 2-propanol/acetonitrile, 60:40 v/v) to the pellet in CHCl₃. Pipette and disperse repeatedly to mix.
4. Spin the solution containing lipids in CHCl₃ and 9-AA at 16,000 x g for 30 s, and then deposit the supernatant as droplets of 0.35 µL (three replicates for each sample) on the MALDI target (sample plate) to be analyzed ('dried droplet' deposition method).
5. Let the droplets air dry at least for 10-15 min.

3. Lipid analysis by MALDI-TOF/MS

1. Acquire mass spectra of samples in triplicates on a MALDI-TOF mass spectrometer.
2. After calibration with lipid standards (see Table of Materials), set the analyses in the negative ion mode and optimize the detection m/z range from 200 Th to 2,000 Th for small molecule analysis.
3. Keep the laser fluence 5% above the threshold (of CL and MLCL) to have a S/N (at least 2).
4. Acquire spectra in reflector mode using delayed pulsed extraction. For each mass spectrum, average 2,000 single laser shots (sum of 4 x 500). Apply gated matrix suppression to 400 Th to prevent detector saturation.

4. How to calculate the (MLCL + CLi)/CLm ratio

1. Run the MALDI-TOF/MS instrument software (see Table of Materials) to analyze the acquired spectra.
2. Using the Open command, open the Spectrum Browser dialog that allows selecting and loading the spectrum of interest.
3. On the menu bar, click on the icons Smooth Mass Spectrum and Subtract Mass Spectrum Baseline.
4. Click on File > Export > Mass Spectrum and choose the ASCII format for exporting the spectrum as a two-column table with pairs of data points: m/z (x) and intensity (y). Copy and paste the coordinates in a spreadsheet program (*.xls).
5. Repeat steps 4.2 to 4.4 for the triplicates of each analyzed sample, pasting the coordinates in the same spreadsheet program file as shown in Figure 1 (Step 1).
6. Following the x-ranges shown in Figure 1 (Step 2) for each species listed, calculate the sum of y₁, y₂, and y₃ values (triplicates) by the SUM function to get the peak area (Figure 1; Step 3).
7. Perform the average of the triplicates area values by AVERAGE function (Figure 1; Step 3).
8. Place the average area values for each species in the column as shown in Figure 1 (Step 2).
9. In order to consider only the first isotopologue of the CLm 72:7 species as in²⁸, calculate the isotopic correction for the overlapping between the M + 2 isotopologue of the CLm 72:8 and the monoisotopic peak of CLm 72:7 as shown in Figure 1 (Step 4).
10. Finally, calculate the (MLCL + CLi)/CLm ratio as shown in Figure 1 (Step 5).

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-diagnosed^{2,6. 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...}

Materials

Name	Company	Catalog Number	Comments
1,1′,2,2′-tetratetradecanoyl cardiolipin	Avanti Polar Lipids	750332	Lipid standard for MALDI-TOF calibration
1,1′2,2′-tetra- (9Z-octadecenoyl) cardiolipin	Avanti Polar Lipids	710335	Lipid standard for MALDI-TOF calibration
1,2-di- (9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine	Avanti Polar Lipids	878130	Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-sn-glycero-3-phosphate	Avanti Polar Lipids	830845	Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-snglycero-3-phospho-(1′-rac-glycerol)	Avanti Polar Lipids	840445	Lipid standard for MALDI-TOF calibration
1,2-ditetradecanoyl-sn-glycero-3-phospho-L-serine	Avanti Polar Lipids	840033	Lipid standard for MALDI-TOF calibration
2-Propanol, ACS reagent, ≥99.5%	Merck Life Science S.r.l.	190764
9-Aminoacridine hemihydrate, 98%	Acros Organics	134410010
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 450,000-650,000	Merck Life Science S.r.l.	31392
Flex Analysis 3.3	Bruker Daltonics		Software
MALDI-TOF mass spectrometer Microflex LRF	Bruker Daltonics
Microsoft Excel	Microsoft Office		Software
OmniPur 10X PBS Liquid Concentrate	Merck 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