Nucleosides/-tide reverse transcriptase inhibitors (NRTI) are included in most antiretroviral treatment (ART) regimens. Exposure to NRTI is associated with a diffuse spectrum of metabolic adverse reactions consistent with mitochondrial toxicity (2
). These side effects were originally thought to be due primarily to the inhibition of mitochondrial DNA polymerase gamma (γ) (3
). Subsequent work suggests that NRTI may cause mitochondrial dysfunction through a variety of mechanisms, which may vary depending on the specific NRTI and cell type (4
). Because the clinical symptoms associated with mitochondrial toxicity are variable and range in severity, there would be substantial clinical utility to a non-invasive diagnostic assay to evaluate mitochondrial toxicity in HIV-infected individuals on ART.
Muscle biopsy is commonly used to diagnose genetic mitochondrial disorders (6
), and adipose biopsies appear sensitive to evaluate antiretroviral (ARV) associated lipodystrophy (7
), but tissue biopsies are not practical for routine monitoring during ART. A variety of non-invasive methods have been developed in an effort to assess mitochondrial toxicity using peripheral blood (9
). The most established approach has been to monitor changes in the amount of mitochondrial DNA (mtDNA) in peripheral blood as measured by quantitative PCR (qPCR) (11
), although these results have been inconsistently associated with metabolic symptoms and/or exposure to NRTI (8
). qPCR amplifies nucleic acid exponentially, which can result in significant inter-assay variability. Despite extensive assay optimization and rigorous standardization, assay variability may be too great for monitoring mitochondria within an individual over time, given that relatively minor changes in the number of mitochondria may influence mitochondrial function. In an effort to improve assessment of mitochondrial toxicity, we developed a method to assess mitochondrial toxicity by quantifying the ratio of mitochondrial to nuclear encoded proteins using flow cytometry (1
). This method was reproducible and potentially measures mitochondria function on a single cell level. However, when we explored the potential utility of the assay the ratio of mitochondrial/nuclear proteins was not associated with exposure to stavudine (d4T) or didanosine (ddI) or symptoms of mitochondrial toxicity such as lipodystrophy. We speculated that the lack of correlation could be because the ratio of nuclear- to mitochondrial-encoded proteins is under homeostatic control such that transcription and translation are regulated in order to maintain a relatively constant ratio of mitochondrial proteins in each cell.
In this study, we compared mitochondrial status in subjects’ peripheral blood mononuclear cells (PBMC) as determined by our flow cytometry-based assay to qPCR, in order to further evaluate the utility of our flow cytometric assay in the detection of NRTI-associated mitochondrial toxicity and help guide future development of diagnostic assays.