Rapid desensitization and uncoupling of human beta-adrenergic receptors in an in vitro model of lactic acidosis

Adrenergic responsiveness in many tissues may be impaired in the presence of lactic acidosis. The purpose of this study was to examine how human neutrophil beta 2-adrenergic receptors may be altered in an in vitro model of lactic acidosis. Receptor coupling was assessed by constructing curves for the competition of isoproterenol with [125I]iodocyanopindolol for beta-adrenergic receptors. Receptors were exposed to control (2 mM lactate, pH 7.4), lactic acidosis (16 mM lactate, pH 7.1), lactate excess (16 mM lactate, pH 7.4), and low pH (2 mM lactate, pH 7.1) conditions. Prior exposure of beta-adrenergic receptors on whole cells to lactic acidosis resulted in a 61% reduction in isoproterenol-stimulated cAMP accumulation (P less than 0.005). This desensitization was not accompanied by down-regulation. After exposure to lactic acidosis, beta-adrenergic receptors were uncoupled sufficiently that a high affinity state could not be detected. Both lactate excess and low pH were necessary to fully express the desensitization and uncoupling defects. The uncoupling was rapid (40 min) and occurred in cell-free membrane preparations. Thus, in an in vitro model of lactic acidosis, rapid desensitization and uncoupling occur which do not appear to require protein synthesis.     

Interferon-gamma and tumor necrosis factor synergize to induce nitric oxide production and inhibit mitochondrial respiration in vascular smooth muscle cells.

Nitric oxide (NO) is an important signal substance in cell-cell communication and can induce relaxation of blood vessels by activating guanylate cyclase in smooth muscle cells (SMCs). NO is synthesized from L-arginine by the enzyme NO synthase, which is present in endothelial cells. It was recently shown that SMCs may themselves produce NO or an NO-related compound. We have studied NO production and its effects on energy metabolism in cultured rat aortic smooth muscle cells. It was observed that the cytokines, interferon-gamma and tumor necrosis factor-alpha, synergistically induced an arginine-dependent production of NO in these cells. This was associated with an inhibition of complex I (NADH: ubiquinone oxidoreductase) and complex II (succinate: ubiquinone oxidoreductase) activities of the mitochondrial respiratory chain, suggesting that NO blocks mitochondrial respiration in these cells. Lactate accumulated in the media of the cells, implying an increased anaerobic glycolysis, but there was no reduction of viability. An NO-dependent inhibition of mitochondrial respiration and a switch to anaerobic glycolysis would reduce energy production of the SMCs. This would in turn reduce the contractile capacity of the cell and might represent another NO-dependent vasodilatory mechanism. It could be of particular importance in inflammation, since cytokines released by inflammatory cells may induce autocrine NO production in SMCs.     

Risk of fatal and nonfatal lactic acidosis with metformin use in type 2 diabetes mellitus: systematic review and meta-analysis

BACKGROUND: Metformin therapy for type 2 diabetes mellitus has been shown to reduce total mortality rates compared with other antihyperglycemic treatments but is thought to increase the risk of lactic acidosis. The true incidence of fatal and nonfatal lactic acidosis associated with metformin use is not known. METHODS: A comprehensive search was performed to identify all comparative trials or observational cohort studies published between January 1, 1959, and March 31, 2002, that evaluated metformin therapy, alone or in combination with other treatments, for at least 1 month. The incidence of fatal and nonfatal lactic acidosis was recorded as cases per patient-years for metformin treatment and for placebo or other treatments. In a second analysis, lactate levels were measured as a net change from baseline or as mean treatment values for metformin and comparison groups. RESULTS: Pooled data from 194 studies revealed no cases of fatal or nonfatal lactic acidosis in 36 893 patient-years in the metformin group or in 30 109 patients-years in the nonmetformin group. Using Poisson statistics with 95% confidence intervals, the probable upper limit for the true incidence of lactic acidosis in the metformin and nonmetformin groups was 8.1 and 9.9 cases per 100 000 patient-years, respectively. There was no difference in lactate levels for metformin compared with placebo or other nonbiguanide therapies. CONCLUSION: There is no evidence to date that metformin therapy is associated with an increased risk of lactic acidosis or with increased levels of lactate compared with other antihyperglycemic treatments if the drugs are prescribed under study conditions, taking into account contraindications.     

Symptomatic hyperlactataemia: an emerging complication of antiretroviral therapy

BACKGROUND: Fatal lactic acidosis is a serious complication of therapy with nucleoside analogues. OBJECTIVE: To examine symptomatic hyperlactataemia in HIV-infected adults treated with antiretroviral drugs. METHODS: In this prospective study, arterial blood lactate levels were measured in patients presenting with unexplained clinical symptoms. When these levels were high, functional respiratory tests (FRT) were carried out. Liver or muscle biopsies were further proposed. Incidences were calculated by comparison with the entire cohort of patients treated in the department. RESULTS: Fourteen HIV-infected adults treated with antiretroviral drugs were identified with symptomatic hyperlactataemia during a 2-year period follow-up study. The incidence of hyperlactataemia was 0.8% per year but reached 1.2% if only patients treated with a regimen including stavudine were considered. Clinical symptoms included abnormal fatigue, tachycardia, abdominal pain, weight loss, peripheral neuropathy, and more specifically exercise-induced dyspnoea occurring despite effective antiretroviral treatment. FRT showed a metabolic deviation towards anaerobiosis with a high lactate/pyruvate ratio. Ultrastructural mitochondrial abnormalities were seen in all four patients for whom this was examined. There was a marked decrease in complex IV activity in muscle biopsies from four of five patients, consistent with a mitochondrial dysfunction. Evolution was favourable in 13 patients, probably because of an early diagnosis. CONCLUSIONS: Potentially fatal adverse events occurring during antiretroviral treatment may be avoided by close monitoring of clinical signs and blood lactate levels. If other studies confirm that the cumulative long-term toxicity of antiretroviral drugs results from mitochondrial dysfunction, the incidence of hyperlactataemia and its clinical consequences may become more important.     

Neurodegeneration and chronic renal failure in methylmalonic aciduria—A pathophysiological approach

Summary In the last decades the survival of patients with methylmalonic aciduria has been improved. However, the overall outcome of affected patients remains disappointing. The disease course is often complicated by acute life-threatening metabolic crises, which can result in multiple organ failure or even death, resembling primary defects of mitochondrial energy metabolism. Biochemical abnormalities during metabolic derangement, such as metabolic acidosis, ketonaemia/ketonuria, lactic acidosis, hypoglycaemia and hyperammonaemia, suggest mitochondrial dysfunction. In addition, long-term complications such as chronic renal failure and neurological disease are frequently found. Neuropathophysiological studies have focused on various effects caused by accumulation of putatively toxic organic acids, the so-called ‘toxic metabolite’ hypothesis. In previous studies, methylmalonate (MMA) has been considered as the major neurotoxin in methylmalonic aciduria, whereas more recent studies have highlighted a synergistic inhibition of mitochondrial energy metabolism (pyruvate dehydrogenase complex, tricarboxylic acid cycle, respiratory chain, mitochondrial salvage pathway of deoxyribonucleoside triphosphate (dNTP)) induced by propionyl-CoA, 2-methylcitrate and MMA as the key pathomechanism of inherited disorders of propionate metabolism. Intracerebral accumulation of toxic metabolites (‘trapping’ hypothesis’) is considered a biochemical risk factor for neurodegeneration. Secondary effects of mitochondrial dysfunction, such as oxidative stress and impaired mtDNA homeostasis, contribute to pathogenesis of these disorders. The underlying pathomechanisms of chronic renal insufficiency in methylmalonic acidurias are not yet understood. We hypothesize that renal and cerebral pathomechanisms share some similarities, such as an involvement of dicarboxylic acid transport. This review aims to give a comprehensive overview on recent pathomechanistic concepts for methylmalonic acidurias. Competing interests: None declared References to electronic databases: OMIM, ExPASY for EC numbers, KEGG metabolism pathways.     

MERRF/MELAS overlap syndrome due to the m.3291T>C mutation

We report the case of a 19-year-old Chinese female harboring the m.3291T>C mutation in the MT-TL1 gene encoding the mitochondrial transfer RNA for leucine. She presented with a complex phenotype characterized by progressive cerebellar ataxia, frequent myoclonus seizures, recurrent stroke-like episodes, migraine-like headaches with nausea and vomiting, and elevated resting lactate blood level. It is known that the myoclonus epilepsy with ragged-red fibers (MERRF) is characterized by cerebellar ataxia and myoclonus epilepsy, while that the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is characterized by recurrent stroke-like episodes, migraine-like headaches, and elevated resting lactate blood level. So the patient’s clinical manifestations suggest the presence of a MERRF/MELAS overlap syndrome. Muscle biopsy of the patient showed the presence of numerous scattered ragged-red fibers, some cytochrome c oxidase-deficient fibers, and several strongly succinate dehygrogenase-reactive vessels, suggestive of a mitochondrial disorder. Direct sequencing of the complete mitochondrial genome of the proband revealed no mutations other than the T-to-C transition at nucleotide position 3291. Restriction fragment length polymorphism analysis of the proband and her family revealed maternal inheritance of the mutation in a heteroplasmic manner. The analysis of aerobic respiration and glycolysis demonstrated that the fibroblasts from the patient had mitochondrial dysfunction. Our results suggest that the m.3291T>C is pathogenic. This study is the first to describe the m.3291T>C mutation in association with the MERRF/MELAS overlap syndrome.     

Brain acidosis

Brain tissue acidosis is a result of either an increase in tissue PCO2 or an accumulation of acids produced by metabolism. Severe hypercapnia (arterial PCO2 around 300 mm Hg) may cause a fall in tissue pH to around 6.6 without any deterioration of the cerebral energy state or morphologic evidence of irreversible cell damage. In severe ischemia and tissue hypoxia, anaerobic glycolysis leads to lactic acid accumulation. This is aggravated by hyperglycemia and by a (trickling) residual blood flow. Under such circumstances lactate concentration in the tissue may increase to levels above 20 to 25 mumol/g (tissue wet weight), causing a decrease in pH to around 6.0. If lactic acidosis during ischemia or hypoxia reaches these excessive levels, metabolic and functional restitution is severely hampered upon subsequent recirculation and reoxygenation. In these circumstances cell morphology shows signs of irreversible damage. Conversely there is less damage if severe tissue lactic acidosis can be hindered. The deleterious effect of excessive lactic acidosis may be related to an influence on the following: synthesis and degradation of cellular constituents; mitochondrial function; cell volume control; postischemic blood flow; and stimulation of pathologic free radical reactions. Possibilities for therapeutic interventions include the avoidance of hyperglycemia, inhibition of glycolysis, and measures for increasing the buffer capacity of the brain.     

Increased lactate production follows loss of mitochondrial membrane potential during apoptosis of human leukaemia cells

Acute tumour-lysis syndrome (ATLS) is a frequently fatal complication after cytoreductive leukaemia therapy. Lactic acidosis is associated with ATLS and its extent is correlated with the severity of ATLS. In the course of cytoreductive therapy, apoptosis is induced in tumour cells, which results in loss of mitochondrial function. We hypothesize that loss of mitochondrial function leads to compensatory glycolysis, which is the main cause of lactate accumulation and acidosis. We tested this hypothesis using the model of glucocorticoid-induced apoptosis in the human acute lymphoblastic leukaemia cell line CCRF-CEM. After induction of glucocorticoid-induced apoptosis, a biphasic course of lactate production was observed. Prior to the onset of apoptosis, i.e. prior to the loss of membrane potential, lactate production was reduced. However, subsequent to loss of mitochondrial membrane potential a massive increase in lactate production was observed (15.5 +/- 0.5 versus 10.17 +/- 0.09 mmol/10(6) cells, P = 0.001). We also demonstrated that inhibition of respiratory chain activity by antimycin A resulted in excess lactate production. In the model cell line used, conditional bcl-2 expression delayed glucocorticoid-induced apoptosis by protecting against loss of mitochondrial membrane potential; bcl-2 expression delayed the increase in lactate production and had no effect on the pre-apoptotic drop in lactate production. Apoptosis-induced lactate production was also observed in other cell lines (HL60, THP1 and OPM2) with various cytotoxic agents [doxorubicin, gemcitabine and vumon (VM26)]. Thus, the data suggest that lactate acidosis can be caused by apoptotic loss of mitochondrial function and massive apoptosis of a tumour mass via lactic acidosis may be the essential pathological event in ATLS.     

Lactic Acidosis: A Rare Oncological Emergency in Solid Tumors at Presentation

Lactic acidosis is a potentially life-threatening complication characterized by accumulation of blood lactate resulting in low arterial pH. The majority of lactic acidosis in malignancies are reported in association with hematologic malignancies. It may result from an imbalance between lactate production and hepatic lactate utilization, but the exact pathophysiology is far more complex than what we can fathom from current micromolecular studies. We report a case of a 71-year-old male with metastatic lung cancer presenting with fatal lactic acidosis in the absence of liver involvement. Review of the literature reveals only 27 reported cases of solid tumors presenting with lactic acidosis, of which nearly all of them had extensive liver metastasis. Patients were treated with aggressive fluid resuscitation, bicarbonate administration and hemodialysis, but the only effective treatment modality was early aggressive chemotherapy initiation.     

Lactic acidosis associated with standard dose linezolid in a kidney recipient with impaired renal function

Severe lactic acidosis, a mitochondrial toxicity caused by the recommended standard dosage of linezolid (LZD), may occur in patients with impaired renal function. We describe an adult male who underwent kidney transplantation with stably impaired renal function, severe dyspnea, and abdominal discomfort. He received a standard oral dose of LZD (600 mg twice daily) and azithromycin for three weeks with a reduced immunosuppressant dose due to pulmonary non-tuberculosis mycobacterial infection. He was alert and afebrile, with a blood pressure of 140/60 mmHg. Pertinent laboratory data showed: pH 7.12, PaCO2 13.6 mmHg; HCO3- 4.3 mmol/L and serum lactate 18.4 mmol/L. His trough serum LZD concentration reached toxic levels (21.4 μg/mL). With hemodialysis, his clinical symptoms improved, with a decline in serum LZD (9.8μg/mL) and lactate (3.2 mmol/L). Chronic standard dose LZD in patients with impaired renal function can lead to life-threatening lactic acidosis, especially in coexisting conditions that reduce LZD metabolism.     

Lactic acidosis in biguanide-treated diabetics

Summary The paper presents an analysis of clinical symptoms, signs and laboratory data of 330 diabetic patients who developed lactic acidosis after having been treated with biguanides (phenformin, buformin, metformin). From the review of the literature an attempt is made to find special features that predisposed patients to develop lactic acidosis such as accompanying illnesses and additional medications, to describe the course of illness and also the factors that influenced the prognosis. Of the patients that developed lactic acidosis 50.3% died. These patients were older, they suffered more frequently from cardiovascular shock, their acidosis was more severe, the whole blood lactate concentration was higher, and the degree of renal insufficiency was more advanced. From our observations we conclude that the treatment of diabetes mellitus with biguanides should be reserved for specially selected patients.     

Lactic acidosis associated with stavudine administration: a report of five cases.

Type "B" lactic acidosis has been described in patients receiving the nucleoside analogs zidovudine, didanosine, and fialuridine. Lactic acidosis has also been described in 4 patients receiving combination therapy with stavudine and lamivudine. We describe the development of chronic type "B" lactic acidosis in 3 patients receiving stavudine as a single agent and in 2 patients receiving combination therapy with stavudine and either lamivudine or delavirdine, a nonnucleoside analog. All patients presented with abdominal pain, vomiting, and hepatic steatosis. Other signs of mitochondrial toxicity included pancreatitis and myopathy (2 cases). The mean duration of stavudine therapy was 9.4 months, and the mean observed peak lactate level+/-SD was 10.3+/-5 mmol/L. After discontinuation of stavudine treatment, lactic acidosis improved in 4 patients after 4-60 weeks, and 1 patient died. Evaluations for other causes of lactic acidosis, including hypoxemia, malignancy, sepsis, and cardiogenic shock, were negative.     

Melatonin prevents age-related mitochondrial dysfunction in rat brain via cardiolipin protection

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Complex I of the mitochondrial respiration chain is an important site of ROS production and hence a potential contributor to brain functional changes with aging. Appropriate antioxidant strategies could be particularly useful to limit this ROS production and associated mitochondrial dysfunction. Melatonin has been shown to possess antioxidant properties and to reduce oxidant events in brain aging. The mechanism underlying this protective effect of melatonin is not well established. In the present study, we examined the effects of long-term treatment of aged rats with melatonin on various parameters related to mitochondrial bioenergetics in brain tissue. After isolation of mitochondria from control, aged, and melatonin-treated young and aged rats, various bioenergetic parameters were evaluated such as complex I activity, rates of state 3 respiration, mitochondrial hydrogen peroxide (H2O2) production, and membrane potential. The mitochondrial content of normal and oxidized cardiolipin was also evaluated. We found that all these mitochondrial parameters were significantly altered with aging, and that melatonin treatment completely prevented these age-related alterations. These effects appear to be due, at least in part, to melatonin's ability to preserve the content and structural integrity of cardiolipin molecules, which play a pivotal role in mitochondrial bioenergetics. The melatonin's ability to prevent complex I dysfunction and cardiolipin peroxidation was also demonstrated by in vitro experiments on brain mitochondria treated with tert-butyl hydroperoxide. In summary, this study documents a decline of mitochondrial bioenergetic functions in brain with aging and the beneficial effect of melatonin.     

Cardioprotection and mitochondrial S-nitrosation: effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in cardiac ischemia-reperfusion injury

Mitochondrial dysfunction is a key pathologic event in cardiac ischemia-reperfusion (IR) injury, and protection of mitochondrial function is a potential mechanism underlying ischemic preconditioning (IPC). Acknowledging the role of nitric oxide (NO()) in IPC, it was hypothesized that mitochondrial protein S-nitrosation may be a cardioprotective mechanism. The reagent S-nitroso-2-mercaptopropionyl-glycine (SNO-MPG) was therefore developed to enhance mitochondrial S-nitrosation and elicit cardioprotection. Within cardiomyocytes, mitochondrial proteins were effectively S-nitrosated by SNO-MPG. Consistent with the recent discovery of mitochondrial complex I as an S-nitrosation target, SNO-MPG inhibited complex I activity and cardiomyocyte respiration. The latter effect was insensitive to the NO() scavenger c-PTIO, indicating no role for NO()-mediated complex IV inhibition. A cardioprotective role for reversible complex I inhibition has been proposed, and consistent with this SNO-MPG protected cardiomyocytes from simulated IR injury. Further supporting a cardioprotective role for endogenous mitochondrial S-nitrosothiols, patterns of protein S-nitrosation were similar in mitochondria isolated from Langendorff perfused hearts subjected to IPC, and mitochondria or cells treated with SNO-MPG. The functional recovery of perfused hearts from IR injury was also improved under conditions which stabilized endogenous S-nitrosothiols (i.e. dark), or by pre-ischemic administration of SNO-MPG. Mitochondria isolated from SNO-MPG-treated hearts at the end of ischemia exhibited improved Ca(2+) handling and lower ROS generation. Overall these data suggest that mitochondrial S-nitrosation and complex I inhibition constitute a protective signaling pathway that is amenable to pharmacologic augmentation.     

Evaluation of sodium acetate as a source of alkali therapy in an experimental aerobic model of lactic acidosis due to decreased pyruvate oxidation

An “in vitro” model of one type of lactic acidosis was produced in rat hemi-diaphragms with inhibitors of pyruvate oxidation. In order to obtain this inhibition in the absence of hypoxia, two actions were sought; inhibiting the mitochondrial pyruvate transporter and lowering the rate of pyruvate diffusion into these mitochondria. α-Cyano-3-hydroxy cinnamate (CNCM) was utilized because it is a specific inhibitor of the mitochondrial pyruvate transporter. Aminooxyacetate (AOA) was employed because it leads indirectly to inhibition of the entry of cytoplasmic reducing power into mitochondria. As a result of the addition of this latter compound, pyruvate levels fell and this should decrease the rate of pyruvate diffusion into the mitochondria. Glucose was the only substrate provided to this tissue and its entry into the cells was promoted by insulin. The oxidation of U-¹⁴C glucose to ¹⁴CO₂ was significantly reduced in the presence of CNCM and AOA, presumably reflecting the inhibition of pyruvate oxidation. Under these conditions, lactate accumulated and pyruvate fell; however, there was a significant accumulation of lactate plus pyruvate during the incubation period. This “in vitro” lactic acidosis was markedly diminished when acetate was also present. These results are consistent with the hypothesis that provision of an alternate substrate to the TCA cycle for ATP synthesis could lead to a decreased rate of glycolysis and thereby to a decreased rate of lactic acid accumulation in this “in vitro” model of lactic acidosis.     

Glucose and lactate kinetics in children with severe malaria

Children with severe malaria often present with lactic acidosis and hypoglycemia. Although both complications independently predict mortality, mechanisms underlying their development are poorly understood. To study these metabolic derangements we sequentially allocated 21 children with falciparum malaria and capillary lactate concentrations of 5 mmol/L or more to receive either quinine or artesunate as antimalarial therapy, and dichloroacetate or saline placebo for lactic acidosis. We then administered a primed infusion (90 min) of L-[3-13C1]sodium lactate and D-[6,6-D2]glucose to determine the kinetics of these substrates. The mean (SD) glucose disposal rate in all patients was 56 (16) micromol/kg x min, and the geometric mean (range) lactate disposal rate was 100 (66-177) micromol/kg x min. Glucose and lactate disposal rates were positively correlated (r = 0.62; P = 0.005). Artesunate was associated with faster parasite clearance, lower insulin/glucose ratios, and higher glucose disposal rates than quinine. Lactate disposal was positively correlated with plasma lactate concentrations (r = 0.66; P = 0.002) and time to recovery from coma (r = 0.82; P < 0.001; n = 15). Basal lactate disposal rates increased with dichloroacetate treatment. Elevated glucose turnover in severe malaria mainly results from enhanced anaerobic glycolysis. Quinine differs from artesunate in its effects on glucose kinetics. Increased lactate production is the most important determinant of lactic acidosis.     

A case risk study of lactic acidosis risk by metformin use in type 2 diabetes mellitus tuberculosis coinfection patients

Metformin (MET) has possibilities to be utilized as an adjunct of tuberculosis (TB) therapy for controlling the growth of Mycobacterium tuberculosis (M. tuberculosis). MET enhances the production of mitochondrial reactive oxygen species and facilitates phagosome–lysosome fusion; those mechanism are important in M. tuberculosis elimination. Moreover, MET-associated lactic acidosis (MALA) needs to be considered and the incidence of MALA in patients with type 2 DM–TB coinfection remains unknown. This result contributes much to our understanding about the clinical effect of MET use in type 2 DM–TB coinfection.For the purpose of understanding the MET effect as an adjuvant therapy in TB therapy and insulin simultaneous therapy, an observational clinical study was done in type 2 DM newly TB coinfection outpatients at Surabaya Paru Hospital. Patients were divided into two groups. First group was MET group, in which the patients were given MET accompanying insulin and TB treatment regimens, the golden standard therapy of DM–TB coinfection. MET therapy was given for at least 2 months. Second group was non-MET group, in which the patients were given insulin and TB treatment regimens. The lactate levels in both groups were measured after 2 months.Among 42 participants, there was no case of lactic acidosis during this study period. Data were normally distributed; thus, we continued analysis of the difference using paired T-test with 95% confidence. There was no difference in lactate levels (p = 0.396) after MET therapy compared to non-MET group.In this study involving patients with TB pulmonary diseases, there is neither evidence that MET therapy induced lactic acidosis event nor that it increased lactate blood level. Thus, we concluded that MET use in type 2 DM–TB coinfection did not induce lactic acidosis.