Detection of brain specific cardiolipins in plasma after experimental pediatric head injury

Abstract

Cardiolipin (CL) is a mitochondria-specific phospholipid that is central to maintenance and regulation of mitochondrial bioenergetic and metabolic functions. CL molecular species display great tissue variation with brain exhibiting a distinct, highly diverse CL population. We recently showed that the appearance of unique brain-type CLs in plasma could serve as a brain-specific marker of mitochondrial/tissue injury in patients after cardiac arrest. Mitochondrial dysfunction has been increasingly implicated as a critical mechanism underlying the pathogenesis of traumatic brain injury (TBI). Therefore, we hypothesized that unique, brain-specific CL species from the injured brain are released to the peripheral circulation after TBI. To test this hypothesis, we performed a high-resolution mass spectrometry based phospholipidomics analysis of post-natal day (PND)17 rat brain and plasma after controlled cortical impact. We found a time-dependent increase in plasma CLs after TBI including the aforementioned brain-specific CL species early after injury, whereas CLs were significantly decreased in the injured brain. Compositional and quantitative correlational analysis suggested a possible release of CL into the systemic circulation following TBI. The identification of brain-type CLs in systemic circulation may indicate underlying mitochondrial dysfunction/loss after TBI. They may have potential as pharmacodynamics response biomarkers for targeted therapies.

Keywords: Brain-specific cardiolipin; Mitochondrial dysfunction; Phospholipidomics; Plasma biomarker.

Figure 1.

Phospholipidome of pediatric rat brain. (A) Chromatogram of phospholipid classes from pediatric rat brain. Spectra showing different class of phospholipids (B) and cardiolipin (C). (D) Bar graph showing number of species identified in each phopsholipid class. (E) Pie chart showing the distribution of a given phospholipid quantity in each class. (F) Heat map showing the amount of different molecular species of major phospholipids except cardiolipin in brain. PA: phosphatidic acid, PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PG: phosphatidylglycerol, PS: phosphatidylserine, CL: cardiolipin, LPG: lyso-PG, LPI: lyso-PI, LPE: lyso-PE, LPS: lyso-PS, LPC: lyso-PC, mCL: monolyso-CL.

Figure 2.

Heat map showing the changes in the brain phospholipidome after CCI. Row Z-score depicts the normalized distribution of CL levels across all samples [Z-score = (Sample value − mean)/SD]. Z-score of 0 is marked by black cells and indicates that the sample value is identical to mean value. Z-score of +3 and −3 are marked bright yellow and by bright blue cells and they indicate that the sample value is 3 standard deviations above or below the mean, respectively. Brain CL levels were lower at 4h and 24h while monolyso-CL and lyso-PL were higher at 4h and 24h after CCI vs. naïve. The lowest brain CLs levels were observed at 4h after injury.

Figure 3.

Phospholipidome of PND17 rat plasma and its changes after traumatic brain injury. Pie charts showing the distribution of number (A) and amount (B) of phospholipid species in plasma. (C) Score plot of orthogonal value vs predictive value from the OPLS-DA model. D) Heat map showing the amount of different species of cardiolipins identififed in plasma in naïve and TBI rats at 4 h and 24 h after injury. Individual species of CL are shown as CL(XX:YY), where XX represent total acyl carbons and YY represent total double bonds. Row Z-score depicts the normalized distribution of CL levels across all samples. [Z-score = (Sample value − mean)/SD]. Z-score of 0 is marked by black cells and indicates that the sample value is identical to mean value. Z-score of +2 and −2 are marked bright red and by bright green cells and they indicate that the sample value is 2 standard deviations above or below the mean, respectively. E) Scatter line plot showing the temporal changes in brain specific cardiolipins in plasma.

Figure 4.

Bar graph showing the changes in the amount of brain cardiolipins vs plasma cardiolipins after TBI. Values are Mean ± SD.