Lipoatrophy and Mitochondrial DNA Assays

In 1999 Dr. Kees Brinkman of the Department of Internal Medicine, Amsterdam, hypothesized that nucleoside analogue reverse transcriptase inhibitors (NRTI) might play an important role in the pathogenesis of lipodystrophy with mitochondrial dysfunction through the inhibition of DNA polymerase gamma. Following is a summary of an editorial, published in the January 3, 2005 issue of AIDS, on the role of mitochondrial DNA assays in detecting mitochondrial toxicity.

Dr. Brinkman begins by stating that recent studies have indeed shown that NRTI, in particular stavudine (d4T; Zerit), are clearly associated with the development of lipoatrophy over time. Recently a prospective study comparing tenofovir (Viread) with stavudine, endorsed these earlier findings. In addition it was shown that especially in patients with exposure to stavudine, serious depletion of mitochondrial DNA (mtDNA) occurred, as well as mitochondrial dysfunction. Finally, in three prospectively conducted studies, Dr. Brinkman notes that a moderate increase of peripheral subcutaneous fat was observed, when patients suffering from lipoatrophy switched away from stavudine.

In the January 3, 2005 issue of AIDS, McComsey et al. further elaborate on one of these switching studies (TARHEEL), providing the link between these clinical observations and the earlier observed stavudine-induced mtDNA depletion. In a substudy on 16 patients, they demonstrate in adipose tissue the restoration of depleted mtDNA after stavudine was switched to abacavir (Ziagen), as well as some improvement in mitochondrial function. Furthermore, writes Dr. Brinkman, they add further proof to earlier observations that increased apoptosis might be the final pathway into lipoatrophy, which decreased after stavudine was changed.

Studies like these are difficult to perform: material is obtained by invasive procedures and especially in prospective studies, this approach requires motivated collaboration of the patients involved. Furthermore, samples are very often too small to check for the texture of the tissue or to perform extra (duplicate) tests. Especially in patients with lipoatrophy, one might easily have an overrepresentation of connective tissue, since most adipocytes might have disappeared by then.

To overcome this tissue sampling problem, many investigators have used more accessible tissue: blood. Studies have already been published, evaluating the effects of NRTI on mtDNA in peripheral blood mononuclear cells (PBMC) and expensive assays have been commercialized and advocated for routine patient care.

However in a recent evaluation among laboratories, the same samples showed considerable variance of 96% (non-log transformed values) in measuring mtDNA content. Furthermore, as was discussed for adipose tissue, PBMC also form a heterogeneous population of different cells (B- and T-lymphocytes, monocytes etc.), all in different stages of activation. Since the kinetics of mtDNA and mitochondrial proteins in these different cell-types are unknown, simply measuring mtDNA content in PBMC, without performing the appropriate checks for homogeneity, will too easily lead to conflicting results. In fact, McComsey et al. found some increase of mtDNA content in PBMC after switching stavudine for abacavir, while Hoy et al. were unable to find such an improvement in a similar switching study.

Finally, untreated HIV-infected patients have been found to have 'lower mtDNA' content in PBMC than healthy controls and spontaneous improvement of mtDNA in PBMC to pretreatment (or higher) levels was noted without any interruption of the previously mtDNA-depleting antiretroviral drug combination.

Dr. Brinkman concludes, “These reports underscore the need for controlling samples both for cell-type as well as for activation status before conclusions can be made.”

 “For the future, only studies that analyze drug toxicity in detail at the level of the representative tissue involved will finally teach us the definite etiology of lipoatrophy.”

01/21/05

Reference
K Brinkman. Lipoatrophy and mitochondrial DNA assays: see all, know all? (Editorial Comment). AIDS 19(1): 91-92. January 3, 2005.