– Nils Venhoff and Ulrich A. Walker –
Two years after the introduction of PIs into the armamentarium of ART, reports of HIV-infected individuals experiencing clinically relevant changes in body metabolism began to surface. These metabolic symptoms were initially summarized under the term “lipodystrophy” (Carr 1998). Nowadays this lipodystrophy syndrome is understood as the result of overlapping yet distinct effects of the different drug components in ART. The main pathogenetic mechanism through which nucleoside analogs are thought to contribute to metabolic changes and organ toxicities is mitochondrial toxicity (Brinkman 1999).
Pathogenesis of mitochondrial toxicity
NRTIs are prodrugs (Kakuda 2000) – they require activation in the cell through phosphorylation before they are able to inhibit their target, HIV reverse transcriptase. In addition to impairing the HIV replication machinery, the NRTI-triphosphates also inhibit a human polymerase called gamma-polymerase, which is responsible for the replication of mitochondrial DNA (mtDNA), a small circular molecule normally present in multiple copies in each mitochondrion and in hundreds of copies in most human cells. Thus, the inhibition of gamma-polymerase by NRTIs leads to a decline (depletion) in mtDNA content (Lewis 2003). The biological task of mtDNA is to encode for enzyme subunits of the respiratory chain, which is located in the inner mitochondrial membrane. Therefore, by causing mtDNA depletion, NRTIs also lead to a defect in the respiratory chain function.
An intact respiratory chain is the prerequisite for numerous metabolic pathways. The main task of the respiratory chain is to oxidatively synthesize ATP, our chemical currency of energy. In addition, the respiratory chain consumes NADH and FADH as end products of fatty acid oxidation. This explains the micro- or macrovesicular accumulation of intracellular triglycerides, which often accompanies mitochondrial toxicity. Last but not least, a normal respiratory function is also essential for the synthesis of DNA, because the de novo synthesis of pyrimidine nucleosides depends on an enzyme located in the inner mitochondrial membrane. This enzyme is called dihydroorotate dehydrogenase (DHODH) (Löffler 1997). The clinical implications of this are detailed below.
The onset of mitochondrial toxicity follows several principles (Walker 2002a):
1. Mitochondrial toxicity is concentration-dependent. High NRTI concentrations cause a more pronounced mtDNA depletion compared to low concentrations. The clinical dosing of some nucleoside analogs is close to the limit of tolerance with respect to mitochondrial toxicity.
2. Mitochondrial toxicity is time dependent and develops with prolonged NRTI exposure. Changes in mitochondrial metabolism are observed only if the amount of mtDNA depletion exceeds a certain threshold. As a consequence, long-term NRTI exposure may lead to mitochondrial effects despite relatively low NRTI concentrations and the onset of toxicity is typically not observed in the first few months of ART.
3. There are significant differences in the relative potencies of nucleoside and nucleotide analogs in their ability to interact with gamma-polymerase. The hierarchy of gamma-polymerase inhibition for the active NRTI metabolites has been determined as follows: zalcitabine (ddC, HIVID®) > didanosine (ddI, Videx®) > stavudine (d4T, Zerit®) > lamivudine (3TC, Epivir®) ≥ abacavir (ABC, Ziagen®) ≥ tenofovir (TDF, Viread®) ≥ emtricitabine (FTC, Emtriva®).
4. Zidovudine (AZT, Retrovir®) may be peculiar because its active triphosphate is only a weak inhibitor of gamma-polymerase. However, another mechanism can explain how zidovudine might cause mtDNA depletion independent from gamma-polymerase inhibition. AZT is an inhibitor of mitochondrial thymidine kinase type 2 (TK2), and, as such, interferes with the synthesis of natural pyrimidine nucleotides, thus potentially impairing the formation of mtDNA (McKee 2004). Indeed, inborn defects of TK2 are known to cause mtDNA depletion in human muscle tissue (Saada 2001). It has also been demonstrated that AZT can be non-enzymatically converted into d4T within the body, at least within some cells (Becher 2003, Bonora 2004).
5. Mitochondrial toxicity is tissue-specific. Tissue specificity is explained by the fact that the uptake of the NRTI prodrugs into cells and their mitochondria as well as activation by phosphorylation may be different in different cell types.
6. There may be additive or synergistic mitochondrial toxicities if two or more NRTIs are used in combination.
7. Data suggest that mitochondrial transcription may also be impaired without mtDNA alterations (Galluzzi 2005, Mallon 2005). However, the mechanism and clinical significance of this are not yet understood.
MtDNA depletion may manifest clinically in one or several target tissues (Figure 1). In the liver mitochondrial toxicity is associated with increased lipid deposits, resulting in micro or macrovesicular steatosis. Steatosis may be accompanied by elevated liver transaminases. Such steatohepatitis may progress to liver failure and lactic acidosis, a potentially fatal, but fortunately rare complication.
Although steatohepatitis and lactic acidosis were already described in the early 90s in patients receiving didanosine monotherapy (Lambert 1990), mitochondrial liver toxicity is now observed with treatment with all NRTIs that have a relatively strong potential to inhibit gamma-polymerase, especially with the so called “d-drugs” ddI, d4T (and formerly, ddC). Liver complications have also been described with AZT. It has been demonstrated in the hepatic tissue of HIV patients that each of the d-drugs leads to a time dependent mtDNA depletion. On electron microscopy, morphologically abnormal mitochondria were observed.
Figure 1: Organ manifestations of mitochondrial toxicity.
A typical complication of mitochondrial toxicity is an elevation in serum lactate. Such hyperlactatemia was more frequently described with prolonged d4T treatment (Carr 2000, Saint-Marc 1999), especially when combined with ddI. The toxicity of ddI is also increased through interactions with ribavirin and hydroxyurea. The significance of asymptomatic hyperlactatemia is unclear. When elevated lactate levels are associated with symptoms, these are often non-specific such as nausea, right upper quadrant abdominal tenderness or myalgias. In the majority of cases, levels of bicarbonate and the anion gap (Na+ – (HCO3– + Cl–)) are normal, although liver transaminases are mildly increased in the majority of cases (Lonergan 2000). Therefore, the diagnosis relies on the logistically more cumbersome direct determination of serum lactate. In order to avoid artifacts, venous blood must be drawn without the use of a tourniquet from resting patients. The blood needs to be collected in fluoride tubes and transported to the laboratory on ice for immediate analysis. Non-mitochondrial causes must also be considered in the differential diagnosis of lactic acidosis (Table 1) and underlying organ toxicities should be looked for. The incidence of lactic acidosis is low; it was estimated as 1 per 1000 patient years in NRTI exposed patients (Imhof 2005). In South Africa, much higher incidence rates (10.6 cases per 1,000 patient years) have been reported (Bolhaar 2007).
Mitochondrial myopathy in antiretrovirally treated HIV+ patients was first described with high dose AZT (Arnaudo 1991). Skeletal muscle weakness may manifest under dynamic or static exercise. The serum CK is often normal or only minimally elevated. Muscle histology helps to distinguish this form of NRTI toxicity from HIV myopathy, which may also occur simultaneously. On histochemical examination, the muscle fibers of the former are frequently negative for cytochrome c-oxidase and carry ultrastructurally abnormal mitochondria, whereas those of the latter are typically infiltrated by CD8-positive T lymphocytes. Exercise testing may detect a low lactate threshold and a reduced lactate clearance, but in clinical practice these changes are difficult to distinguish from lack of aerobic exercise (detraining).
In a murine model of cardiomyopathy, nine weeks of oral AZT and ddC induced a mitochondrial lesions with mtDNA depletion, diminished respiratory chain function and ultrastructural abnormalities of mitochondria (Balcarek 2010).
Table 1: Causes of hyperlactatemia/ lactic acidosis.
|Type A lactic acidosis||Type B lactic acidosis|
|(Tissue hypoxia)ShockCarbon monoxide poisoningHeart failure||(Other mechanisms)Thiamine deficiencyAlkalosis (pH >7.6)Epilepsy
Adrenalin (iatrogenic, endogenous)
Neoplasm (lymphoma, solid tumors)
Intoxication (nitroprusside, methanol, methylene glycol, salicylates)
Rare enzyme deficiencies
Prolonged treatment with d-drugs can also lead to a predominantly symmetrical, sensory and distal polyneuropathy of the lower extremities (Moyle 1998, Simpson 1995). An elevated serum lactate level can help distinguish this axonal neuropathy from its HIV-associated phenocopy, although in most cases the lactate level is normal. The differential diagnosis may also take into account the fact that the mitochondrial polyneuropathy mostly occurs weeks or months after initiation of the d-drugs. In contrast, the HIV-associated polyneuropathy generally does not worsen and may indeed improve with prolonged antiretroviral treatment. In mice AZT and ddC induce a mitochondrial neurotoxicity with ddC predominantly affecting the peripheral and AZT mainly the central nervous system (Venhoff 2010). This is consistent with findings in patients (Tardieu 2005, Moyle 1998). Although the toxic effects of AZT on the central nervous system have not been studied well in humans, they are plausible because AZT penetrates well into the CNS.
In its more narrow sense, the term lipodystrophy denotes a change in the distribution of body fat. Some individuals affected with lipodystrophy may experience abnormal fat accumulation in some body areas (most commonly in the abdomen or in the dorsocervical region), whereas others may develop fat wasting (Bichat’s fat pad in the cheeks, temporal fat, or subcutaneous fat of the extremities). Both fat accumulation and fat loss may occur simultaneously in the same individuals. Fat wasting (also called lipoatrophy) is partially reversible and generally observed not earlier than one year after the initiation of antiretroviral therapy. In the affected subcutaneous tissue, ultrastructural abnormalities of mitochondria and reduced mtDNA levels have been identified, in particular in subjects treated with d4T (Walker 2002b). In vitro and in vivo analyses of fat cells have also demonstrated diminished intracellular lipids, reduced expression of adipogenic transcription factors (PPAR-gamma and SREBP-1), and increased apoptotic indices. NRTI treatment may also impair some endocrine functions of adipocytes. For example, NRTIs may impair the secretion of adiponectin and through this mechanism may promote insulin resistance. D4T has been identified as a particular risk factor, but other NRTIs such as zidovudine may also contribute. When d4T is replaced by another NRTI, mtDNA levels and apoptotic indices improve (McComsey 2005), along with an objectively measurable, albeit small increase of subcutaneous adipose tissue (McComsey 2004). In contrast, switching away from protease inhibitors did not ameliorate lipoatrophy or adipocyte apoptosis. Taken together, the available data indicate a predominant effect of mitochondrial toxicity in the pathogenesis of lipoatrophy.
Some studies have suggested an effect of NRTIs on the mtDNA levels in blood (Coté 2003, Miro 2003). The functional consequence of such mitochondrial toxicity on lymphocytes is still unknown. In this context, it is important to note that a delayed loss of CD4 and CD8 lymphocytes was observed when ddI plasma levels were increased by co-medication with TDF or when ddI was given to subjects with low body weight (Negredo 2004). Recent in vitro investigations with exposure of mitotically stimulated T lymphocytes to only slightly supratherapeutic concentrations of ddI also detected a substantial mtDNA depletion with a subsequent late onset decline of lymphocyte proliferation and increased apoptosis (Setzer 2005a, Setzer 2005b). Thus, mitochondrial toxicity is the most likely explanation for the late onset decline of lymphocytes observed with ddI. The data suggest that the mitochondrial toxicity of NRTIs on lymphocytes has immunosuppressive properties.
Asymptomatic elevations in serum lipase are not uncommon under ART, but of no value in predicting the onset of pancreatitis (Maxson 1992). The overall frequency of pancreatitis has been calculated as 0.8 cases/100 years of NRTI-containing ART. Clinical pancreatitis is associated with the use of ddI in particular. DdI re-exposure may trigger a relapse and should be avoided. A mitochondrial mechanism has been cited to explain the onset of pancreatitis, but this assumption remains unproven.
Furthermore an elevation of serum urate was observed on therapy with dideoxynucleosides (ddI and d4T). Impaired ATP production as a result of mitochondrial toxicity may increase urate production in the purine nucleotide cycle (Walker 2006b).
After long and controversial discussions, several studies now provide evidence that TDF does cause mitochondrial damage to the kidney. TDF is a nucleotide analog reverse transcriptase inhibitor (Viread®) which has been associated with cases of renal dysfunction, Fanconi’s syndrome and cases of osteomalacia in animals (Tenofovir review team 2001) and patients (Gupta 2008, Wanner 2009). Patients treated with TDF often present with elevated serum alkaline phosphatase and hypophosphatemia due to a diminished renal phosphate resorption (Kinai 2005). Also osteomalacia was observed in patients treated with TDF, especially when combined with lopinavir/r (Parsonage 2005, Wanner 2009). TDF is taken up into the proximal renal tubules by human renal organic anion transporters (hOATs) 1 and 3. Despite the fact that TDF only has a low potency to impair the polymerase gamma, the hOATs may generate high intratubular tenofovir concentrations that then interfere with the replication of mtDNA (Cote 2006). Decreased mtDNA levels have been found in renal biopsies from patients exposed to TDF+ddI, an NRTI combination that is no longer recommended (Cote 2006). In rats, TDF induced an organ-specific nephrotoxicity with mtDNA depletion and tubular dysfunction of mtDNA-encoded respiratory chain subunits (Lebrecht 2009). It is therefore not recommended to use TDF in patients with established renal dysfunction.
The use of AZT to avoid vertical HIV transmission diminishes mtDNA levels in the placenta, as well as in the peripheral cord blood of perinatally exposed newborns (Divi 2004, Shiramizu 2003, Gingelmaier 2009). AZT was found to be incorporated into mtDNA in pregnant monkeys treated with AZT plus 3TC prior to delivery and mtDNA depletion was found in skeletal muscle, heart and brain (Gerschenson 2004) in which perinatally acquired lesions were shown to persist for months after cessation of NRTI exposure in some models.
Mitochondrial symptoms and abnormal cerebral imaging were found at increased frequency in infants perinatally exposed to NRTIs (Blanche 1999, Tardieu 2005). Hyperlactatemia is not infrequently observed and may persist for several months after delivery (Noguera 2003). Other clinical trials in contrast did not detect an increased perinatal risk in association with perinatal AZT prophylaxis although key parameters of mitochondrial dysfunction were not assessed. Long-term follow-up data are urgently needed (Venhoff 2006). The available information however does not justify deviating from the currently recommended strategy to use AZT to prevent vertical HIV transmission as part of a combination therapy for the mother.
There is currently no method to reliably predict the mitochondrial risk of an individual patient. Routine screening of asymptomatic NRTI-treated subjects with lactate levels is not warranted, since elevated lactate levels in asymptomatic subjects are not predictive of clinical mitochondrial toxicity (McComsey 2004). Quantification of mtDNA levels in PBMCs is not reliable. The [13C]methionine breath test can be used to analyze the oxidative capacity of liver mitochondria, but results may be confounded by several factors other than HIV or ART (Sternfeld 2009). Quantifying mtDNA within affected tissues is likely to be more sensitive; however this form of monitoring is invasive and not evaluated with regard to clinical endpoints.
Once symptoms are established, histological examination of a biopsy may contribute to the correct diagnosis. The following findings in tissue biopsies point towards a mitochondrial etiology: ultrastructural abnormalities of mitochondria, diminished histochemical activities of cytochrome c oxidase, the detection of intracellular and more specifically microvesicular steatosis, and the so-called ragged-red fibers.
Treatment & prophylaxis of mitochondrial toxicity
Drug interactions may precipitate mitochondrial symptoms and must be taken into account. The mitochondrial toxicity of ddI for example is augmented through drug interactions with ribavirin, hydroxyurea and allopurinol (Ray 2004). When ddI is combined with TDF, the dose of ddI must be reduced to 250 mg once daily. The thymidine analog brivudine is a herpes virostatic that may sensitize for NRTI-related mitochondrial toxicity because one of its metabolites is an inhibitor of DHODH (see below). Brivudine should therefore not be combined with antiretroviral pyrimidine analogues.
An impairment of mitochondrial metabolism may also result from ibuprofen, valproic acid and acetyl salicylic acid as these agents impair the mitochondrial utilization of fatty acids. Numerous cases have been described in which a life-threatening lactic acidosis was triggered by valproic acid both in HIV-infected patients and in patients with inherited mutations of mtDNA. Acetyl salicylic acid may damage mitochondria and such damage to liver organelles may result in Reye’s syndrome. Amiodarone and tamoxifen also inhibit the mitochondrial synthesis of ATP. Acetaminophen and other drugs impair the antioxidative defense (glutathione) of mitochondria, allowing for their free radical-mediated damage. Aminoglycoside antibiotics and chloramphenicol not only inhibit the protein synthesis of bacteria, but under certain circumstances may also impair the peptide transcription of mitochondria as our bacteria-like endosymbionts. Adefovir and cidofovir are also inhibitors of gamma-polymerase. Alcohol is a mitochondrial toxin and should be avoided. Lastly, TDF nephrotoxicity may be precipitated by coadministation with lopinavir/r (Wanner 2009). Lopinavir/r increases tenofovir serum levels and also may inhibit MDR4, with both mechanisms then contributing to intratubular tenofovir accumulation.
The most important clinical intervention is probably the discontinuation of the NRTI responsible for mitochondrial toxicity. Several studies have demonstrated that switching from stavudine (Zerit®) to a less toxic alternative led to an objective and progressive improvement in lipoatrophy (Martin 2004, McComsey 2004, Moyle 2004). In contrast, a switch from protease inhibitors to NRTIs was not associated with an improvement of lipoatrophy. These findings stress the importance of mitochondrial toxicity in the pathogenesis of fat abnormalities.
When toxic NRTIs can not be avoided, the supplementation of uridine is currently a very promising strategy to treat mitochondrial toxicity. As outlined above, any respiratory chain impairment also results in the inhibition of DHODH, an essential enzyme for the synthesis of uridine and its derived pyrimidines (Figure 2). This decrease in intracellular pyrimidine pools leads to a relative excess of the exogenous pyrimidine nucleoside analogs, with which they compete at gamma-polymerase. A vicious circle is closed and contributes to mtDNA depletion. By supplementing uridine either prophylactically or therapeutically, this depletion may be interrupted, resulting in increased mtDNA levels (Setzer 2008). Indeed, uridine abolishes all the effects of mtDNA depletion in hepatocytes and normalizes lactate production, cell proliferation, the rate of cell death and intracellular steatosis (Walker 2003). In d4T-exposed adipocytes uridine was able to normalize mitochondrial function and lipid metabolism (Walker 2006a). New data indicate that uridine is able to also prevent ddC-induced hepatotoxicity (Lebrecht 2007), and AZT-induced myopathy (Lebrecht 2008), cardiomyopathy and neuropathy (Venhoff 2010) in mice.
The oral substitution of uridine as a pyrimidine precursor is well tolerated by humans, even at high doses (Kelsen 1997, van Groeningen 1986). Mitocnol, a food supplement, was shown to have a more than 8-fold uridine bioavailability over conventional uridine (Venhoff 2005).
Figure 2: Suggested mechanism of mitocnol (NucleomaxXTM) in the prevention and treatment of mitochondrial toxicity.
A randomized placebo-controlled double-blind trial found that mitocnol improved subcutaneous fat in lipoatrophic subjects under continued therapy with d4T or AZT (Sutinen 2007). In a second study mitocnol was found to be efficacious with regard to patient- and physician-assessed lipoatrophy scores (McComsey 2008). ACTG 5229, a prospective, 1:1 randomized, placebo-controlled multicenter trial evaluated the effectiveness of uridine in improving limb fat in HIV-infected individuals with lipoatrophy on a tNRTI-containing regimen (stratified by AZT or d4T). Primary endpoint was change in limb fat from baseline to week 48. Despite a small improvement in limb fat after 24 weeks compared to placebo, the effect was not sustained through 48 weeks of uridine (McComsey 2010). These results dashed the hopes in uridine as a treatment option of tNRTI-associated lipoatrophy.
Mitochondrial steatohepatitis is antagonized by mitocnol in animal models, as well as in HIV-positive patients (Walker 2004, Banasch 2006, Lebrecht 2007). In AZT-induced myopathy in mice mitocnol attenuated mtDNA depletion and muscle atrophy (Lebrecht 2008). Furthermore, mitochondrial cardiomyopathy and neuropathy was successfully prevented by mitocnol in an animal model (Balcarek 2010, Venhoff 2010).
Mitocnol is well tolerated and adverse events have not been observed so far. In one study, a clinically insignificant HDL decline was noted, while in another HDL cholesterol was unchanged (McComsey 2008). There are no known negative interactions of uridine with antiretroviral treatment (Koch 2003, McComsey 2005, Sommadossi 1988, Sutinen 2007). In Europe and North America, mitocnol is available as a dietary supplement called NucleomaxX® and can be acquired in pharmacies and the internet (www.nucleomaxX.com).
With symptomatic hyperlactatemia and with lactic acidosis, NRTIs should be immediately discontinued (Brinkman 2000).
With respect to mtDNA depletion vitamin supplements were not found to be effective either in vitro or in clinical studies (Venhoff 2002, Walker 1995). In animals and humans mitocnol improves hyperlactatemia (Lebrecht 2007, Sutinen 2007). NRTI re-exposure may be possible after normalization of lactate (Lonergan 2003). The supportive treatment of hyperlactatemia and lactic acidosis is summarized in Table 2.
Table 2: Supportive treatment of lactate elevation in HIV-infected patients (non-pregnant adults).
|Lactate 2-5 mmol/L + symptoms||Lactate > 5 mmol/L or lactic acidosis|
|Discontinue mitochondrial toxinsConsider vitamins and|
NucleomaxX® (36g TID on 3 consecutive days/month)
Discontinue NRTIs and all mitochondrial toxinsIntensive care
Maintain hemoglobin >100 g/L
Avoid vasoconstrictive agents
Bicarbonate controversial – 50-100 mmol if pH <7.1
Coenzyme Q10 (100 mg TID)
Vitamin C (1 g TID)
Thiamine (Vit. B1, 100 mg TID)
Riboflavin (Vit. B2, 100 mg QD)
Pyridoxine (Vit. B6, 60 mg QD)
L-acetyl carnitine (1 g TID)
NucleomaxX (36 g TID until lactate <5 mmol/L)
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