Lai et al1 reported in 1991 for the first time a case of severe lactic acidosis and fulminant hepatic failure in an HIV-infected patient on didanosine (ddI) antiretroviral monotherapy. Since then, the introduction of the highly active antiretroviral therapy (HAART) has increased the risk of suffering from secondary effects and several additional reports have confirmed that patients who receive some nucleoside analogues reverse transcriptase inhibitors (NRTIs), particularly ddI and stavudine (d4T), are at increased risk for developing hyperlactatemia and lactic acidosis,2,3 a condition thought to be amongst the most serious adverse effects attributed to HIV and antiretroviral drugs.
Increased blood lactate levels may be associated with a multitude of accompanying and unspecific symptoms including fatigue, weakness, abdominal pain, weight loss, tachycardia, or dyspnea. The clinical presentation of hyperlactatemia is strikingly variable and the severity of symptoms usually shows a good positive correlation with plasma lactate levels. Cohort studies report that 15%-20% of persons/yr who receive a stable HAART regimen develop asymptomatic hyperlactatemia3; these patients show blood lactate levels usually between 2 and 5 mmol/L. Otherwise, 1% of patients/yr develop symptomatic hyperlactatemia, which is usually associated with plasma lactate levels above 5 mmol/L and one or more of the above mentioned manifestations.3 Finally, 0.4-1‰ of patients on HAART/yr present lactic acidosis (blood pH imbalance), which is often associated with higher lactate levels and severe symptomatology, which can lead to death in up to 50% of cases through fulminant hepatic failure.2,3 The prevalence and incidence of hyperlactatemic-related disorders is increasing in developing countries (because of growing access to antiretrovirals) and shows a trend towards reduction in the developed world because of physicians awareness and available routinary lactate measures, although unfortunately it is still a quite frequent and life-threatening event.
Mitochondrial toxicity of antiretroviral drugs, particularly NRTIs, has been postulated to be responsible for the etiopathogesis of many secondary effects of HAART,4,5 including hyperlactatemia. Moreover, HIV itself could extend the mitochondrial adverse effects of antiretrovirals by modulating inflammatory and/or apoptotic cellular mechanisms.6,7 Mitochondria are the center of energy supply in nearly all body cells by coupling ATP synthesis to oxygen consumption through the oxidative phosphorylation (OXPHOS) system. Specifically, hyperlactatemia can be the result of hypoxic atmospheres or mitochondrial dysfunction which drives energy production out of the mitochondria through anaerobic metabolism and lactic acid generation, which acidifies blood through conversion into lactate and consequent proton release. Blood lactate concentration is the result of lactate production through anaerobic glycolysis (in all body cells but especially on skeletal muscle, liver, nervous and lymphoid system) and its plasmatic clearance by gluconeogenic pathways (mainly on the liver and secondary on the kidney). NRTIs inhibit the unique enzyme responsible for mitochondrial DNA (mtDNA) replication (DNA polymerase γ),8-10 increase the number of mtDNA mutations and reduce the number of entire mitochondrial genomes. Because mtDNA encodes for 13 proteins of the OXPHOS system responsible for aerobic energy production, important mtDNA depletions can lead to mitochondrial dysfunction moving energy production towards anaerobic metabolism and lactate production. But the complexity of HAART-induced mitochondrial toxicity is however increasing with the description of alternative mechanisms for mitochondrial lesion by NRTIs in absence of mtDNA depletion11-15 and homeostatic mechanisms able to compensate severe mtDNA depletion and preserve mitochondrial function.16
The hypothetic connection between antiretroviral-mediated mitochondrial toxicity and hyperlactatemia was first reported on liver17-19 and skeletal muscle17,19-23 as mtDNA depletion and/or OXPHOS system dysfunction. These investigations included however a small number of patients, used invasive approaches and, in most of them, the exploration of mitochondria was partial and limited to mtDNA quantification. Additionally, a correlation between mitochondrial parameters and blood lactate levels was lacking. We herein present the replicational, transcriptional, translational, and biochemical mitochondrial analysis of 26 HIV-infected patients under HAART who developed hyperlactatemia with different degree of clinical severity and lactate levels, both during the hyperlactatemic episode and after clinical recovery, to better assess mitochondrial basis of HAART-related hyperlactatemia. We used a noninvasive method since we studied peripheral blood mononuclear cells (PBMCs). These results have been compared with the values found in nonhyperlactatemic HIV-infected patients on HAART (treated), infected but untreated HIV individuals (naive) and noninfected volunteers (healthy controls).
PATIENTS AND METHODS
We studied genetic and biochemical PBMC mitochondrial parameters of 26 consecutive HIV-infected patients on HAART undergoing an hyperlactatemic episode (lactate levels above 2 mmol/L) and after clinical recovery. Patients were recruited during 3 years on the Infectious Diseases department of 4 different Catalan hospitals (Hospital Clinic of Barcelona, Hospital Germans Trias i Pujol of Badalona and Hospital Joan XXIII and Hospital of Sant Pau i Santa Tecla from Tarragona) because of increased lactate levels, sometimes accompanied by clinical symptomatology, or because they presented severe accompanying symptomatology with moderate to high lactate values. Patients were categorized in 3 different clinical forms, according to clinical presentation: 13 were asymptomatic, 8 were symptomatic, and 5 had lactic acidosis. We considered as symptoms of hyperlactatemia: fatigue, weakness, abdominal pain, weight loss, tachycardia, and/or dyspnea, after other causes of disease were conveniently discarded. Lactate levels, immunovirologic parameters and one sample of peripheral blood for mitochondrial studies were obtained on admission and after the clinical recovery of hyperlactatemia. Clinical and antiretroviral histories were obtained from the patients' medical records to be correlated with mitochondrial toxicity results. An extensive work-up was performed to exclude other causes of hyperlactatemia. In most of cases hyperlactatemic episode prompted antiretroviral treatment withdrawal that was exclusively restarted, most of times after changing its composition, after clinical recovery and lactate normalization or because of severe immunovirologic reasons.
We compared the results of these hyperlactatemic patients with respect to 3 control groups of subjects that were consecutively collected during the same period of time in the same participant hospitals: 28 nonhyperlactatemic HIV subjects on HAART (treated), 31 HIV-infected but untreated individuals (naive), and 20 uninfected controls (healthy). Clinical and epidemiological characteristics of included patients and controls are summarized on Table 1. Control group inclusion was made trying to match individual characteristics with those of the hyperlactatemic group of subjects. Treated nonhyperlactatemic patients were matched by sex, age, and time on HIV infection, and time on HAART and time on d-drug treatment (ddI and/or d4T administration) with the hyperlactatemic patients. Naive subjects were matched by sex with hyperlactatemic individuals, but presented statistical significant differences (P < 0.05) with respect to age and time on HIV infection. Healthy volunteers presented differences in terms of age and sex with respect the rest of studied groups. All those differences found among our study groups represent, in most of cases, those found in the general population.
All individuals were informed and signed written consent to be included in this protocol that was approved by the Ethical Committee of the Hospital Clinic of Barcelona.
To avoid confounders of mitochondrial toxicity, those patients taking other potentially toxic drugs for mitochondria (ie, aminoglycosides, linezolid, or statins) were excluded from the study, and those subjects with familiar history of mitochondrial disease.
Mononuclear cells (lymphocytes and monocytes) were isolated by Ficoll density gradient centrifugation24 and we confirmed a platelet count below 25 per PBMC in all patients coming from the different groups suggestive of negligible platelet contamination.
Protein content was measured according to the Bradford protein-dye binding-based method.25 Samples were frozen at −80°C until mitochondrial analysis.
Total DNA was obtained by the standard phenol-chloroform extraction procedure. A fragment of the mitochondrial-encoded ND2 gene and the nuclear-encoded 18S rRNA gene were amplified in duplicate and separately by quantitative real-time PCR using Lightcycler Roche thermocycler (Roche Diagnostics, Mannheim, Germany), as previously reported.26,27 The relative content of mtDNA was expressed as the ratio between mtDNA and nDNA amount (ND2 mtDNA/18S rRNA nDNA content).
Mitochondrial RNA Quantification
Total RNA was obtained by an affinity column-based procedure (Rneasy; Qiagen Sciences, Germantown, MD). RNA was reverse-transcribed to cDNA using random hexamer primers and the real-time PCR reaction used to quantify relative mitochondrial cDNA content was performed using Applied Biosystems technology in an ABI PRISM 7700 sequence detection system (Applied Biosystems Inc., Foster City, CA). Quantification of the mitochondrial encoded cytochrome c oxidase subunit-II (COX-II) mRNA and the nuclear-encoded housekeeping 18S rRNA were performed using the amplification conditions and the primers previously reported.27 The relative content of mitochondrial RNA (mtRNA) was expressed as the ratio between mtRNA and nuclear RNA (nRNA) amount (COX-II mtRNA/18S rRNA nRNA content).
Mitochondrial Protein Synthesis
We assessed mitochondrial protein synthesis of the COX-II subunit (mitochondrially encoded, transcribed, and translated) by western blot immunoanalysis.16,27 This expression was normalized by the content on the mitochondrially located COX-IV subunit (nuclear-encoded and cytoplasmically transcribed and translated) to establish the relative mitochondrial protein expression amount (mtCOX-II/nCOX-IV protein abundance).
Mitochondrial OXPHOS Complexes II, III, and IV (COX) Enzyme Activity
All mitochondrial enzymatic activities were measured spectrophotometrically according to the Rustin et al28 methodology, slightly modified for complex IV measurement in minute amounts of biological samples.29 OXPHOS complex II is completely encoded, transcribed and translated by cytoplasmic machinery, whereas CIII and CIV (COX) complexes are partially encoded, transcribed and translated by mitochondrial means. Specific enzymatic activities were expressed in absolute values as nanomols of synthesized substrate or consumed product per minute and milligram of measured protein (nmol/min/mg protein).
The main outcome was the assessment of genetic or biochemical PBMC's mitochondrial parameter change of HIV-infected patients on HAART undergoing a hyperlactatemic crisis (lactate levels over 2 mmol/L) and after clinical recovery and lactate normalization. As mitochondrial genetics parameters we considered mtDNA and mtRNA content and mitochondrial protein synthesis amount. As mitochondrial biochemical parameters we considered those enzymatic activities which take part of the mitochondrial respiratory chain (complexes II, III, and IV).
Additionally, mitochondrial results of hyperlactatemic patients during and after the episode were compared with respect to 3 control groups: nonhyperlactatemic HIV subjects on HAART (treated), HIV-infected untreated individuals (naive), and uninfected volunteers (healthy).
Results were expressed as mean ± standard error of the mean (SEM) or as percentage with respect to healthy controls, the latter were arbitrarily assigned as 100%. We ascertained the normal distribution of mitochondrial and clinical parameters using the Kolmogorov-Smirnov analysis. Parametric T-test for independent or paired normal-distributed measures (as needed) were used to search for differences and regression analysis was used to find relationship between quantitative parameters. Otherwise, for nonnormal-distributed parameters, the nonparametric test Mann-Whitney was used to search for independent sample differences, Wilcoxon paired rank test for paired comparisons and Spearman's rank coefficient to search for parameter correlation.
A P value of less than 0.05 was considered significant.
Patients suffering from hyperlactatemia on admission had a mean blood lactate value of 3.7 ± 0.6 mmol/L (normal range: 0.8-2 mmol/L). Awareness of early clinical suspicious of hyperlactatemia and routine lactate measurement screening on current HIV clinical management made the lactate level of our patients to be lower with respect to that found in the past or in previous reports,1-3 and most of our patients were, because of that, asymptomatic (13 of the 26 included). All hyperlactatemic patients were reassessed 9.7 ± 1.2 months later, when blood lactate levels were normalized (1.9 ± 0.1, P = 0.01 compared with the baseline values) and they achieved clinical recovery.
Mitochondrial Analyses During Hyperlactatemia and After Clinic Recovery
Hyperlactatemic patients presented all mitochondrial parameters decreased during the hyperlactatemic episode and with respect clinical recovery and lactate normalization (Figs. 1-4). The mitochondrial parameters which showed a most significant improvement after the hyperlactatemic episode resolution were the mitochondrial protein synthesis (P < 0.05) and the OXPHOS enzymatic activities of respiratory complexes III and IV (P < 0.01 and P < 0.001, respectively, Figs. 1, 3, and 4).
Mitochondrial Analyses of Hyperlactatemic Patients Compared With Control Groups
During hyperlactatemia, mtDNA was decreased with respect to the 3 control groups; the comparisons achieved statistical significance with respect to HIV-naive patients (P < 0.05) and healthy persons (P < 0.01; Fig. 2). This decrease was accompanied by a diminished amount on mitochondrial transcription (mtRNA) and translation species amount (mitochondrial proteins; as shown in Figs. 1 and 2), although the statistical significance was only achieved when hyperlactatemic values were compared with healthy people (P = 0.01 and P < 0.05, respectively).
The unique parameter which remained nearly equal in all groups was OXPHOS complex II enzymatic activity (Fig. 3). Complex II is not mtDNA encoded rather is entirely encoded by nuclear DNA and entirely transcribed and translated on cytoplasmic ribosomes, and consequently, is supposed to be conserved. Conversely, OXPHOS complexes III and IV function was significantly decreased compared with HIV naive (P < 0.05 and P < 0.01, respectively) and healthy controls (P < 0.01 and P < 0.05, respectively; Fig. 3).
If we consider the values found on healthy people as 100%, patients developing hyperlactatemia had a remaining content of 52% of mtDNA, 46% of mtRNA amount, 63% of mitochondrial protein expression quantity and 49% and 69%, respectively, of OXPHOS complex III and IV enzymatic activities (Fig. 4).
Correlation Between Mitochondrial and Clinic Parameters
The comparison for all these mitochondrial parameters according to the subtype of hyperlactatemia (asymptomatic, symptomatic, or lactic acidosis) did not render statistical differences (data not shown), maybe because of the reduced statistical power of so small groups. But when we assessed the relationship between mitochondrial disturbances and the severity of hyperlactatemia measured as blood lactate levels we found that such a relationship existed only for the enzymatic activity of OXPHOS complexes III and IV, which were negatively correlated with lactate concentration (P < 0.05 in both cases; Fig. 5), whereas mitochondrial genetic parameters did not (P = 0.99 for mtDNA, P = 0.41 for mtRNA and P = 0.38 for mitochondrial protein expression; data not shown).
We found that HAART-related hyperlactatemia is associated with a decrease in all mitochondrial parameters assessed with respect to control values of healthy people and, in some cases (mtDNA and OXPHOS complexes III and IV), also with respect to naive patients. Nonetheless, although mitochondrial parameters were lower than in HIV-infected patients on HAART with normal lactate, none of these differences achieved statistical significance. Interestingly, although all mitochondrial parameters trend to increase after recovery of the hyperlactatemic episode, only mitochondrial translation and OXPHOS complexes III and IV enzymatic activities significantly increase. In addition, although PBMC have been demonstrated a reliable and noninvasive model to perform mitochondrial studies in hyperlactatemic patients, it is foreseeable we can not discard that mitochondrial deficits are bigger in more energy-dependent tissues or those target centers of lactate homeostasis (liver and muscle). Overall, we believe that our findings support the mitochondrial basis for HIV and HAART-related hyperlactatemia.
Mitochondrial toxicity of antiretroviral drugs has been associated mainly with NRTIs use due to its capacity to inhibit mtDNA replication.4,5 Among dideoxynucleoside analogues, d4T seems to be the most powerful inducer of hyperlactatemia,19,22,23,30,31 albeit toxic effects of other d-drugs has not been discarded. Most of our hyperlactatemic patients were taking d4T, but most remarkable is the great amount of these hyperlactatemic subjects that were receiving d4T in combination with ddI in comparison to those treated patients who did not developed the hyperlactatemic disorder. Current guidelines strongly discourage concomitant administration of d4T and ddI, but most of the studied hyperlactatemic patients were included in 2004, when such antiretroviral combination was quite common.
As mtDNA encodes for mitochondrial OXPHOS components, NRTIs-induced mtDNA depletion would led to mitochondrial function impairment. This hypothesis is supported by our data; the finding of mtDNA depletion during hyperlactatemia is associated with a downstream decay of mitochondrial transcription, translation and function. A striking feature of our study is that during the hyperlactatemic episode, mitochondrial biochemistry abnormalities better correlated with blood lactate levels than mitochondrial genetics. In agreement with this finding, when blood lactate normalized and clinical recovery was achieved, mitochondrial-encoded OXPHOS complex III and IV enzymatic activities, together with the mitochondrial protein expression, significantly recovered, whereas mitochondrial replication and transcription species amount did not.
The mitochondrial hypothesis of HAART toxicity launched by Brinkman and colleagues in 19988 has gained complexity during the last recent years. First, reports show that, even in the absence of mtDNA depletion, NRTIs are able to cause mitochondrial lesion independent to DNA polymerase γ inhibition.11-15 Second, severe mtDNA depletion induced by NRTIs has been reported to be compensated by mitochondrial transcriptional or translational upregulatory homeostatic mechanisms.16 These mechanisms could maintain mitochondrial function on adverse circumstances. However, during hyperlactemia, transcription and translation intermediates were decreased, suggesting lack of upregulatory response. Third, assessment of the mitochondrial function has become essential because it is the expected consequence of the genetic lesion and is, ultimately, the responsible of clinical symptoms. Using this overall approach, it has been shown that the change from a highly mitochondriotoxic HAART to other drug schedules with lower toxic potential for mitochondria is first accompanied with a recovery of mitochondrial functions, even if not net changes in mtDNA content are observed.32,33 This finding suggests that mitochondrial functional recovery antedates the improvement of the genetic lesion or, possibly, that the improvement achieves only one part of the mechanisms disrupted. Both are possible explanations for recovery of mitochondrial function after hyperlactatemia in the absence of substantial mtDNA improvement. But finally we can not discard that mtDNA content of hyperlactatemic patients before the crisis could be so close to the threshold limit value which supports mitochondrial function that the small decay occurred during the hyperlactatemic episode, even not statistically significant, could cross this critical value leading to impaired mitochondrial function. Whatever the explanation is, mitochondrial dysfunction is the final determinant to drive energy production out of the mitochondria towards the cytoplasmatic anaerobic glycolytic pathway responsible of lactate production. All studied patients presented such an increase in blood lactate levels and decreased mitochondrial parameters, but each one of them presented one or more of these parameters especially altered. Consequently with other toxic or genetic mitochondrial diseases that correlate with increased lactate levels but have different mitochondrial parameter impairment etiology, mitochondrial dysfunction of HIV and antiretroviral-induced hyperlactatemia could stand at different genetic, biochemical or synthetic mitochondrial levels, and increased lactate production would just be the common consequence of final mitochondrial impairment.
Although HAART-related hyperlactatemia can be developed in uninfected patients exclusively exposed to antiretroviral therapy,34 in chronically treated patients' scenario we can not forget the HIV and mitochondria interactions. HIV is able to cause mitochondrial diffuse genetic5,7 and functional7,35 lesion by itself that could be mediated by indirect inflammatory or apoptotic mechanisms. The mitochondrial damage present in HIV-infected patients on HAART that underwent an hyperlactatemic crisis could be due to the summatory effect of both HIV-induced damage (also present in naive patients) and mitochondrial toxicity of antiretroviral drugs (also found in nonhyperlactatemic asymptomatic subjects). All these additive adverse effects on mitochondrial function could not be exclusively related to interference of mtDNA replication.
At the present time we can not completely eradicate HIV infection, but we can minimize HIV secondary effects, like mitochondrial lesion, by reducing viral load through antiretroviral administration.36 Current guidelines advice the beginning of antiretroviral therapy before it did in the past and one of the benefits of early HIV suppression could be avoiding HIV-induced mitochondrial damage. But we have to take care choosing which drugs to use, at which doses and which antiretroviral combinations can be administered together, because the management of all these parameters will also determinate accumulative and chronic mitochondrial damage and future development of adverse clinical events with mitochondrial basis, like hyperlactatemia. Clinicians must be aware of any early sign or symptom of coming toxicities and therapy change could be welcome not only after an hyperlactatemic crisis, but also previously to its development. Although we demonstrate that mitochondrial recovery is possible after an hyperlactatemic episode, it is essential to prevent secondary effects of HAART better than managing them. Once hyperlactatemia is developed, early management of all disturbances and normalization of lactate and acidemia, will help in mitochondrial and clinical recovery achievement.
Currently available information about HAART-related adverse event etiology has moved antiretroviral guidelines to less potent mitotoxic drug administration, which has fortunately reduced associated mitochondrial damage and derived adverse events, like hyperlactatemia. In developed countries these strategies consist on reducing antiretroviral doses, changing HAART-schedules to nucleoside-sparing regimens or guiding patients to structured-treatment interruptions,33,37-40 but scarce work has been done to evaluate strategies which actively reverts mitochondrial induced damage, even in the context of concomitant antiretroviral administration, as mitochondrial drug therapy.41 Further studies should be addressed to assess how to prevent or correct mitochondrial function in HIV-infected and HAART-treated symptomatic or asymptomatic patients but also to find premature toxicity markers that would allow us avoiding adverse effects of chronic HIV infection and treatment. The performance of mitochondrial assays in noninvasive and easy-obtaining samples (as mononuclear cells) based on measuring functional parameters and non-exclusively limited on measuring mtDNA content could be a useful tool for these screenings.
We are in debt with many people who has contributed to the present work by providing us their valuable help. Among them: Sònia López, Mireia Nicolàs, Jordi Guallart, Maria Larrousse, Ágat León, Ángel Ballesteros, Joaquim Peraire, Sergi Veloso, Consuelo Viladés, and Elisabet Deig.
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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
HIV; HAART; hyperlactatemia; mitochondria; mitochondrial toxicity; mitochondrial function/dysfunction