Lipoatrophy and hyperlactatemia are well-recognized complications of antiretroviral therapy that have been linked to nucleoside reverse transcriptase inhibitor (NRTI)–induced mitochondrial toxicity. 1–4 Measurement of venous lactate levels is currently the only noninvasive marker of mitochondrial toxicity available for use in clinical practice. Unfortunately, lactate levels are nonspecific and insensitive for the detection of early mitochondrial dysfunction. Studies have found prevalence of hyperlactatemia (elevated blood lactate) in up to 36% of largely asymptomatic NRTI-treated subjects, but the significance of asymptomatic hyperlactatemia is debated. In fact, longitudinal studies have yet to show any prog nostic significance of asymptomatic hyperlactatemia. In contrast to the ill-defined asymptomatic form, symptomatic hyperlactatemia is well recognized, and its consequences, if untreated, could be devastating. Significant mitochondrial abnormalities and mitochondrial DNA (mtDNA) depletion have been found in the liver and muscle of subjects with symptomatic mitochondrial toxicity. 5–7
Lipoatrophy or peripheral fat wasting is a newly recognized complication of HIV therapy. Likely reflecting the lack of standardized definitions for the features of this syndrome, estimates of its prevalence among HIV-infected adults range from 2% to 84%.
Recent studies have illustrated the key role of NRTIs in the generation of lipoatrophy. Several groups showed a significant decrease of mtDNA content in the fat of subjects with lipoatrophy. 7–10 Contrary to what has been demonstrated for adipose tissue mtDNA in the setting of lipoatrophy and for liver and muscle mtDNA in symptomatic hyperlactatemia, however, there appears to be no depletion in blood mtDNA of subjects with lipoatrophy and/or symptomatic hyperlactatemia according to most publications. 10–12 These are unfortunate findings, because biopsies are invasive and could not be performed routinely in clinical practice. Therefore, there remains a need for an accurate noninvasive marker of mitochondrial toxicity.
The exact mechanism of NRTI-induced mitochondrial dysfunction remains unclear. One mechanism for the tissue damage induced by NRTI-associated mitochondrial dysfunction is the accumulation of reactive oxygen free radicals, which are normally neutralized by functional mitochondria. 13–16 Zidovudine (ZDV) exposure is able to cause significant mtDNA damage and a decrease in glutathione levels. 14 The addition of N-acetyl cysteine was able to prevent the fall in glutathione and the damage in mtDNA. 14
ZDV has been shown to increase 8-oxo-7,8-dihydro 2-deoxyguanosine (8-oxo-dG), a marker of oxidative damage to DNA. 15 Antioxidants (vitamins C and E) resulted in a significant decrease in 8-oxo-dG and prevented morphologic changes of muscle mitochondria in ZDV-treated mice. 15 Similar changes of oxidative markers were seen with stavudine therapy, and these changes were also prevented by concomitant treatment with antioxidants. 16 In inherited mitochondrial diseases, several studies had suggested a close association with oxidative stress and mtDNA abnormalities. 17,18 In fact, reactive oxygen species could be destructive to both DNA and proteins such that they could alter enzymes in the electron transport chain, directly or indirectly through mtDNA.
A series of bioactive prostaglandin F2–like compounds (termed F2-isoprostanes) were described by Morrow et al. 19 The F2-isoprostanes are derived from arachidonic acid, which undergoes peroxidation catalyzed by free radicals to yield arachidonyl radical intermediates, which are then transformed to a series of prostaglandin F2–like compounds. Levels of free F2-isoprostanes increase dramatically in animal models of oxidant injury. 20 Measurement of F2-isoprostanes has been found to be a useful marker of oxidative damage and lipid peroxidation in a number of diseases, including neurodegenerative disorders and atherosclerosis. In fact, quantification of F2-isoprostanes is widely considered to be the most accurate method to measure oxidant stress in vivo. 21,22
We evaluated the plasma levels of F2-isoprostanes in a group of NRTI-treated subjects with or without sustained hyperlactatemia. Blood was drawn into tubes containing EDTA and immediately centrifuged to separate the plasma. An aliquot of plasma was then removed and immediately stored at −70°C until measurement of F2-isoprostanes was performed. The F2-isoprostanes are quantified in biologic fluids after Sep-Pak and TLC purification as pentafluorobenzyl ester, trimethylsilyl ether derivatives utilizing stable isotope dilution techniques with deuterated 15-F2t-IsoP (Cayman Chemical, Ann Arbor, MI) as an internal standard. The precision of the assay is ±4%, the accuracy ±95%, and interassay variability is less than 8%. 23 Normal levels of plasma F2-isoprostanes are 0.035 ± 0.012 ng/mL. 23
Overall, 59 subjects were included in this analysis: 16 subjects with sustained hyperlactatemia and 43 with normal lactate. Both groups were identified through an ongoing prospective longitudinal study of the natural history of hyperlactatemia and its relationship with lactic acidosis and other metabolic complications. This study uses strict criteria for collection and processing of lactate levels. 24 Preliminary results of this longitudinal lactate study on the first 250 enrolled subjects had shown a low prevalence of 4% hyperlactatemia at the time of study entry, 24 supporting the fact that strict methods for collection and processing are needed to avoid falsely elevated lactate levels. Overall, this longitudinal study included 396 randomly selected subjects from our clinic population and was able to identify a total of 16 subjects who exhibited sustained hyperlactatemia (lactate >2.0 mmol/L) between October 23, 2000 and January 28, 2003. This was defined as elevation of more than 50% of the lactate determinations, with, at a minimum, 2 elevated levels. As a control, we used a group of 43 subjects with normal lactate levels at all time points of the longitudinal study (median of 6 normal lactate values per patient). Four of the 16 patients with sustained hyperlactatemia had symptoms consistent with hyperlactatemia. These were nausea (3 patients), fatigue (3 patients), abdominal pain (3 patients), anorexia (3 patients), increased liver transaminases (3 patients), weight loss (3 patients), dyspnea (3 patients), and diarrhea (2 patients). All these symptoms were of recent onset (within the last 2 weeks prior to the most recent lactate measurement) and of moderate to severe intensity. Only 1 patient had metabolic acidosis (bicarbonate of 10). All 4 patients had lactate levels drawn while remaining on NRTI-containing antiretrovirals. The F2-isoprostane measurements were obtained during the course of the longitudinal lactate study previously mentioned. Specifically, specimens from the 4 subjects with symptomatic hyperlactatemia were obtained during the symptomatic phase; those from the 14 subjects with sustained asymptomatic hyperlactatemia were drawn at the end of the longitudinal lactate study, after at least 2 elevated lactate levels. In subjects with persistently normal lactate, the blood was drawn at the end of the longitudinal lactate study (ie, after a median of 6 consecutive normal lactate levels per patient). The detailed results of the longitudinal lactate study will be published elsewhere.
Participants in this study were further stratified by the presence or absence of clinical lipoatrophy. For the purpose of this study, lipoatrophy was defined as loss of fat in at least 2 of the following areas: face, arms, legs, and buttocks. This had to be confirmed by an experienced registered dietitian and/or investigator.
Table 1 summarizes the baseline characteristics and the laboratory indices in the group of subjects with sustained hyperlactatemia and in the subjects with persistently normal lactate. Both groups differed by lactate levels, smoking, duration of HIV disease, and duration of abacavir therapy. Using the Wilcoxon signed rank test, there was no difference in F2-isoprostane levels between subjects with normal lactate and subjects with sustained asymptomatic hyperlactatemia (0.053 ± 0.027 vs. 0.053 ± 0.021, respectively; P = 0.977).
The 59 patients were further stratified by the presence or absence of clinical lipoatrophy. Table 2 summarizes the baseline characteristics and the laboratory indices in the lipoatrophy and the no-lipoatrophy groups. Using the Wilcoxon signed rank test, plasma F2-isoprostane was significantly higher in the lipoatrophy group compared with the no-lipoatrophy group (0.060 ± 0.025 vs. 0.0420 ± 0.02, respectively; P = 0.02). These groups were matched for all factors that are known to influence oxidative stress, except for current smoking, which was higher in the no-lipoatrophy group. Overall, there were 34 patients with lipoatrophy and 21 without lipoatrophy. The analysis of F2-isoprostane levels between the lipoatrophy and no-lipoatrophy groups had excluded the 4 subjects with symptomatic hyperlactatemia, because this is a significant confounding variable that could by itself lead to increased oxidative stress. In fact, the mean F2-isoprostane level in these 4 symptomatic subjects was 0.082 ± 0.021 (and was significantly higher than that in subjects without symptomatic hyperlactatemia). Two of these 4 subjects had lipoatrophy, and 2 did not.
No difference in levels of F2-isoprostanes was found between subjects with normal lactate levels and subjects with sustained asymptomatic hyperlactatemia. The groups were matched for parameters that may increase oxidative stress, except for having significantly more smokers in the sustained hyperlactatemia group. This difference in the number of smokers, if anything, should have led to an elevation of F2-isoprostane levels in the hyperlactatemia group. 25
In this study, most of the confounding parameters that may increase oxidative stress were also similar between the lipoatrophy group and the control group. These included duration of NRTI therapy, age, duration of HIV disease, alcohol use, and concurrent use of vitamins and antioxidants. Smoking was more prevalent in the no-lipoatrophy group, which makes the results even more significant, because smoking itself can lead to elevated levels of oxidative markers 25; this could have underestimated the positive association that was found between F2-isoprostane levels and lipoatrophy. Studies to date have shown that the use of antioxidants (in particular, vitamins C and E) may lead to a decrease in F2-isoprostane levels. 23 Importantly, the use of antioxidants was matched between the lipoatrophy and no-lipoatrophy groups.
We were able to show a significant increase in circulating products of lipid peroxidation, F2-isoprostanes, in subjects with clinically defined lipoatrophy. Studies had shown an increase in oxidative stress (increase in lipid peroxides and serum malondialdehyde) in HIV-infected patients, but no studies had previously investigated the correlation of these markers with NRTI-induced toxicities or with lipoatrophy in particular. Whether supplementation of antioxidant vitamins or other approaches can reduce oxidative stress and improve lipoatrophy is unknown.
The results showed that the levels of F2-isoprostanes were significantly higher in patients with clinical lipoatrophy than in a well-matched NRTI-treated group without lipoatrophy. Interestingly, there was no significant difference in F2-isoprostane levels between patients with persistently normal lactate and those who exhibited a sustained asymptomatic increase in lactate. This could be explained by the yet unclear significance of asymptomatic hyperlactatemia, even in a setting like ours, where lactate levels were obtained with close attention to the method of collection and processing. To date, no single study has shown any predictive value of elevated lactate levels in asymptomatic subjects 26,27; therefore, most experts do not recommend routine monitoring of lactate in asymptomatic subjects. In contrast, the 4 subjects with symptomatic hyperlactatemia/lactic acidosis had a significant increase in their F2-isoprostanes.
These findings represent the first evidence of the occurrence of lipid oxidation in patients with lipoatrophy and symptomatic hyperlactatemia, and they corroborate the hypothesis that free radicals are linked to the pathogenesis of NRTI-linked mitochondrial dysfunction. This should encourage the investigation of antioxidants in lipoatrophy.
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