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Mejia et al. J Transl Genet Genom 2024;8:216-24 https://dx.doi.org/10.20517/jtgg.2024.11 Page 218

MATERIALS AND METHODS
Epstein-Barr virus transformed BTHS B lymphoblasts from 4- to 9-year-old males and male age-matched
control lymphoblasts were graciously provided by Dr. Richard Kelley, Kennedy Kreiger Institute, Baltimore,
MD., and obtained from Coreill Institute (Camden, NJ) and were cultured as previously described .
[9]
14
[ C]Creatine was obtained from American Radiochemicals Inc. (Burnaby, BC, Canada, Catalog number
ARC 0176-50 µCi). Ecolite scintillant was obtained from ICN Biochemicals (Montreal, Quebec, Canada).
All other chemicals were of American Chemical Society (ASC) grade and obtained from either Sigma
Aldrich Canada Ltd. (Oakville, ON, Canada) or Thermo Fisher Scientific (Carlsbad, CA, USA).

Electrospray ionization mass spectrometry (ESI-MS) coupled with high-performance liquid
chromatography (HPLC) mass spectrometry of cardiolipin (CL) and monolysocardiolipin (MLCL) from
cell lysates was performed as described . Mitochondrial fractions were isolated using the mitochondrial
[18]
isolation kit from Abcam (Toronto, ON, Canada, Catalog number ab110170). Mitochondrial protein
content was determined using the M protein assay kit (Mississauga, ON, Canada). For Blue-Native
polyacrylamide gel electrophoresis (BN-PAGE) analysis, mitochondrial protein (80 µg) was treated with
[9]
0.2% digitonin and then separated on a 3%-12% gradient gel as described . Immunoblot analysis of the gel
was performed using anti-PKCδ antibody (1:1,000) (Abcam, Toronto, ON, Canada) as described . PKCδ
[19]
was visualized using the Amersham Enhanced Chemiluminescence Western blotting detection system
(VWR, Mississauga, ON, Canada). Band intensity was quantified using Image J software. Citrate synthase
activity was measured using the citrate synthase assay kit (Sigma-Aldrich, Oakville, ON, Canada, Catalog
number CS0720).

Cells were cultured in RPMI 1,640 medium containing 10% fetal bovine serum and 1% antimycotic and
antibiotic solution and incubated at 37 °C in 5% CO until used. Cells were incubated with 2 mL medium
2
containing 0.1 µM [ C]Creatine (specific activity 50-60 mCi/mmol) for up to 60 min. At the indicated time
14
points, the medium was removed and cells washed twice with 2 mL of ice-cold PBS. The PBS was removed
and 2 mL of methanol:water (1:1 v/v) was added. The cells were harvested using a rubber policeman and put
into test tubes. The mixture was vortexed, and a 50 μL aliquot was taken for protein determination and a
50 μL aliquot taken for determination of radioactivity. Data are expressed as mean ± standard deviation of
the mean. Comparisons between control and BTHS patient lymphoblasts were performed by unpaired, two-
tailed Student’s t-test. A probability value of P < 0.05 was considered significant.

RESULTS
All major molecular species of CL were significantly reduced in BTHS lymphoblasts compared to age-
matched control lymphoblasts [Figure 1A]. This reduction in CL molecular species was accompanied by a
general, but not significant, increase in most major MLCL species. In contrast, a > 20-fold increase
(P < 0.01) in trioleoyl-MLCL [mass/charge (m/z) 1192] molecular species was observed in BTHS
lymphoblasts compared to age-matched control lymphoblasts [Figure 1B].

BTHS patient lymphoblasts exhibited abnormally increased mitochondrial mass . To confirm this,
[7,8]
mitochondrial fractions were prepared and citrate synthase activity determined. Citrate synthase activity
was elevated 20% (P < 0.05) in BTHS lymphoblasts compared to age-matched control cells [Figure 1C].
Thus, the reduction in CL, increase in MLCL and MLCL/CL ratio, and increase in citrate synthase activity
were consistent with that observed in BTHS patient B lymphoblast cells.