Correlation of HMPAO uptake with live single cell synchrotron mid-Infrared spectromicroscopy in inherited mitochondrial disease.

Objectives HMPAO uptake is decreased in genetic mitochondrial disease despite normal to increased cerebral perfusion. To understand this phenomenon we subjected fibroblasts from patients with genetic mitochondrial disease to oxidative stress and examined HMPAO uptake. We now wish to correlate the redox spectral fingerprints obtained by single cell FTIR spectromicroscopy with HMPAO uptake.

Methods Cells underwent 30 minutes of oxidative stress with tert-butylhydroperoxide (t-BHP, 0.2 mM). Cells were then washed and pelleted for HMPAO uptake assay. 5 uL of HMPAO was added to samples of 0.5 to 2 million cells and incubated for 30 minutes at 37^oC. Cells were then washed and counted. Live single cells were loaded, physiologically maintained and underwent t-BHP stress while interrogated via FTIR spectromicroscopy. The spectral markers of lipid and protein oxidation are in the non-peptide carbonyl vibration region with vibrational frequencies between 1690 and 1750 cm^-1, and in the peptide carbonyl region between 1600 and 1690 cm^-1.

Results SURF-1 and Cox-10 fibroblasts had lower HMPAO uptake compared to control. After t-BHP stress SURF-1 and controls had 55% and 43.9% decreases in uptake, respectively while Cox-10 fell 181%. Analysis of the FTIR data revealed that without stress, the spectra associated with lipid and protein oxidation were higher both in SURF-1 and Cox-10 cells relative to normal fibroblasts. With t-BHP treatment, the FTIR signal intensity in the non-peptide carbonyl region increased for both SURF-1 and Cox-10 relative to the controls. Furthermore, the signal intensity associated with lipid oxidation in Cox-10 was almost 50% higher than in SURF-1.

Conclusions Decreases in HMPAO uptake of fibroblasts from patients with inherited mitochondrial disease correlate with increased rates of protein and lipid oxidation as well as decreases in glutathione.

Research Support Gift funds from the Mitochondrial Disease Institute at Stanford