By Dr Seul Kee Byeon and Dr Akhilesh Pandey

An elevated ratio of monolysocardiolipin to cardiolipin is a hallmark feature of Barth syndrome, a genetic disorder caused by mutations in the TAFAZZIN gene. Liquid chromatography-tandem mass spectrometry can be used as a clinical laboratory test to calculate the ratios of monolysocardiolipins to cardiolipin species for an early diagnosis of Barth syndrome.

Barth syndrome

Barth syndrome (BTHS; OMIM# 302060 [1]) is an inborn error of phospholipid metabolism resulting from mutations in the TAFAZZIN gene, located at Xq28 [2,3]. First described in 1983, patients with BTHS are primarily males and they often present with dilated cardiomyopathy, neutropenia, skeletal myopathy, growth delay, fatigue and organic aciduria [2,4]. Affecting all ethnic groups, BTHS has a prevalence of 1 in 300 000–400 000 live births in the United States of America. To date, more than 200 different pathogenic variants in BTHS have been identified [2,5].

Cardiolipins and BTHS

TAFAZZIN encodes a phospholipid transacylase enzyme, tafazzin, crucially involved in the remodelling pathway of a lipid known as cardiolipin (CL). Unlike typical phospholipids with two fatty acyl chains, CL is a unique subclass characterized by four fatty acyl chains. Synthesized within the inner mitochondrial membrane, CL is closely linked with mitochondrial functions and related disorders [6]. Initially, nascent CL is produced from cytidine diphosphate-diacylglycerol and phosphatidylglycerol, after which fatty acyl chains can be cleaved from nascent CL by phospholipase to form monolysocardiolipin (MLCL). Subsequently, new side chains are added to generate remodelled CL, involving multiple enzymes such as tafazzin, MLCL acyltransferase 1, and lysocardiolipin acyl-
transferase 1 (Fig. 1). Within this CL remodelling pathway, tafazzin plays a pivotal role and loss-of-function mutations in TAFAZZIN, observed in BTHS, lead to MLCL accumulation and a concurrent decrease in remodelled CL.

Diagnosis of Barth syndrome

The typical approach to diagnosing BTHS involves assessing clinical symptoms and conducting laboratory evaluations, followed by genetic testing to confirm mutations in the TAFAZZIN gene. Various laboratory examinations are employed, including blood tests to evaluate neutrophil levels, cardiac assessments to gauge cardiac function, and biochemical tests to measure levels of 3-methyl-glutaconic acid (3-MGC) and 3-methylglutaric acid (3-MGA) [7,8]. However, the sensitivity and specificity of these biochemical tests are limited because BTHS manifests a wide range of phenotypic variations, with some patients not exhibiting increased levels of 3-MGC or 3-MGA or neutropenia [8,9]. A more conclusive biochemical test aiding in the diagnosis of BTHS is a dried bloodspot (DBS)-based test, which measures the ratio of two specific lipid species: 52:2-MLCL and 72:8-CL [10,11].

Figure 1. Remodelling pathway of cardiolipin
Nascent cardiolipin goes through cycles of deacylation to generate monolysocardiolipin, which then progresses to mature cardiolipin via cycles of reacylation in the cardiolipin remodelling pathway. ALCAT2, Acyl-CoA:lysocardiolipin acyltransferase-2; MLCLAT1, monolysocardiolipin acyltransferase 1d; PLA2, phospholipase A2

Liquid chromatography-tandem mass spectrometry for CL analysis

Mass spectrometry (MS) was initially introduced by J.J. Thomson over a century ago as a tool to measure the mass-to-charge ratio of ions. In the subsequent decades, numerous technological advancements have propelled it to become a routine analytical technique in clinical laboratories. Newborn screening, toxicology, and endo-
crine laboratories have actively embraced MS-based platforms for clinical testing [12]. Chromatographic separation preceding MS analysis can enhance the sensitivity and specificity of target analytes. Among various chromatographic methods, liquid chromatography (LC) and gas chromatography (GC) are commonly employed. Unlike GC, where analytes require derivatization to become volatile, LC does not necessitate sample derivatization. Consequently, liquid chromatography-tandem MS (LC-MS/MS) is primarily used for analysing non-volatile lipids such as CL. CL comprises a highly diverse lipidome characterized by fatty acyl side chains containing 14 to 22 carbons with 0 to 6 double bonds. The use of LC enables the separation of lipids based on their physicochemical properties, thereby optimizing detection by MS and various species of MLCL and CL have been characterized using LC-MS/MS [13,14,15].

A new CL assay to diagnose Barth syndrome

In the USA, there is currently no blood-based clinical test available to measure MLCL and CL, despite around 10 new patients being diagnosed with BTHS each year in the country. Because MLCL and CL species can be present in different combinations of fatty acyl chains, we are in the process of developing a DBS-based LC-MS/MS assay capable of measuring various species of MLCL and CL. This multiplexed approach has the potential to facilitate early diag-
nosis of BTHS and provide the overview of MLCL and CL profiles.

Methods

1. Lipid extraction
Eight 1/8-inch diameter circles of DBS are punched out and Cardiolipin Mix I (Avanti Polar Lipids) is added as internal standards. MLCL and CL lipids are extracted from DBS using the lipid extraction described previously [11]. Briefly, chloroform:methanol (1:1, v/v) is added as extraction solvent, followed by bath sonication. The supernatant is dried under nitrogen and reconstituted in methanol.

2. LC-MS/MS
A new targeted method has been developed using a QTRAP 6500+ mass spectrometer (SCIEX) coupled to a Vanquish™ Horizon ultra-high-performance liquid chromatography system (Thermo Fisher). Lipids are separated on a C18 column in reversed-phase LC gradient. MLCL and CL species are selected based on a prior discovery analysis of DBS lipids where lipids were annotated based on the precursor ion and its corresponding MS/MS spectra. As MLCL and CL are anionic lipids, the detection of lipids was operated in negative ion mode. Deprotonated adducts of MLCL and CL are used as precursor ions and carboxylate anions corresponding to fatty acyl chains are used as productions.

Figure 2. Multiple reaction monitoring chromatograms of (A) internal standards, (B) dried bloodspot (DBS) monolysocardiolipins (MLCLs) and (C) DBS cardiolipins (CLs)
Based on pooled samples of 8 unaffected controls and 8 patients with Barth syndrome (BTHS), altered levels of MLCL and CL species were observed.

MLCL and CL profiles are altered in Barth syndrome

MLCLs and CLs from pooled samples of patients with BTHS are compared to those from pooled unaffected controls using the new LC-MS/MS method. In addition to 52:2-MLCL and 72:8-CL, several other species of MLCL and CL were targeted in this highly multiplexed method. Lipids were separated using reversed-phase LC gradient where lipids elute in increasing order of hydrophobicity. Cardiolipin Mix I used as internal standards contain four species of non-endogenous CL species; 57:4-CL, 61:1-CL, 80:4-CL and 86:4-CL. The numbers preceding the lipid class (e.g. CL) refer to total number of carbons (e.g. 57) and double bonds (e.g. 4) of all the chains.  Species that are more hydrophobic, such as 86:4-CL (green; Fig. 2A), elute at later retention time than other species.

MLCLs contain three fatty acyl chains while CLs contain four fatty acyl chains, making MLCLs less hydrophobic than CLs. As expected, MLCL species elute at earlier retention times (6–8 minutes) then CL species (8–11 minutes), as shown in Figure 1B,C. Each peak in Figure 2B,C corresponds to distinct MLCL and CL species bearing varying total numbers of carbons and double bonds. Although the MS signals of the internal standards added to pooled controls and pooled patient samples were comparable (Fig. 2A), significant differences in the endogenous profiles of the MLCL and CL lipidome were observed between patients and controls. Patients exhibited pronounced elevation of MLCL species and reduction of CL species compared to controls (Fig. 2B,C).

Increased ratios of MLCLs to CLs are observed in Barth syndrome

A total of 34 MLCL and CL species, varying in the length of fatty acyl chains and double bonds, were observed, and various ratios of MLCL/CL species were computed. As MLCL accumulated and CL decreased concurrently, calculating the MLCL/CL ratio could amplify these differences in patients and enhance the differentiation between controls and patients. In addition to 52:2-MLCL/72:8-CL, the ratios of several other MLCL/CL species showed significant elevation in patients. Table 1 summarizes some exemplary lipid ratios. Although these MLCL/CL ratios in unaffected controls were below 0.1, those in patients with BTHS exhibited ratios higher than 10.

Conclusions

The use of DBS in clinical tests offers practical advantages in terms of sample collection, transport, storage, and cost-effectiveness, making it an ideal tool for diagnostic and research purposes. We are presently in the process of completing method development. Once finalized, this highly multiplexed LC-MS/MS assay can be used to examine alterations in multiple species of MLCLs and CLs from DBS, assisting in the initial identification and monitoring of patients with BTHS.

The author

Seul Kee Byeon *1 PhD and Akhilesh Pandey1,2 MD, PhD
1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester MN, USA
2 Center for Individualized Medicine, Mayo Clinic, Rochester MN, USA

* Corresponding author
Email: Byeon.seulkee@mayo.edu

Table 1. Ratios of MLCL / CL species calculated from BTHS patient and control DBSs

References
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The author

Seul Kee Byeon *1 PhD and Akhilesh Pandey1,2 MD, PhD
1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester MN, USA
2 Center for Individualized Medicine, Mayo Clinic, Rochester MN, USA

* Corresponding author
Email: Byeon.seulkee@mayo.edu