Molecular and biochemical alterations in tubular epithelial cells of patients with isolated methylmalonic aciduria.

Methylmalonic acidurias (MMAurias) are a group of inherited disorders in the catabolism of branched-chain amino acids, odd-chain fatty acids and cholesterol caused by complete or partial deficiency of methylmalonyl-CoA mutase (mut(0) and mut(-) subtype respectively) and by defects in the metabolism of its cofactor 5'-deoxyadenosylcobalamin (cblA, cblB or cblD variant 2 type). A long-term complication found in patients with mut(0) and cblB variant is chronic tubulointerstitial nephritis. The underlying pathomechanism has remained unknown. We established an in vitro model of tubular epithelial cells from patient urine (hTEC; 9 controls, 5 mut(0), 1 cblB). In all human tubular epithelial cell (hTEC) lines we found specific tubular markers (AQP1, UMOD, AQP2). Patient cells showed disturbance of energy metabolism in glycolysis, mitochondrial respiratory chain and Krebs cycle in concert with increased reactive oxygen species (ROS) formation. Electron micrographs indicated increased autophagosome production and endoplasmic reticulum stress, which was supported by positive acridine orange staining and elevated levels of LC3 II, P62 and pIRE1. Screening mTOR signaling revealed a release of inhibition of autophagy. Patient hTEC produced and secreted elevated amounts of the pro-inflammatory cytokine IL8, which was highly correlated with the acridine orange staining. Summarizing, hTEC of MMAuria patients are characterized by disturbed energy metabolism and ROS production that lead to increased autophagy and IL8 secretion.

TNF-α mediates mitochondrial uncoupling and enhances ROS-dependent cell migration via NF-κB activation in liver cells.

Development of hepatocellular carcinoma (HCC) is accompanied by a continuous increase in reactive oxygen species (ROS) levels. To investigate the primary source of ROS in liver cells, we used tumor necrosis factor-alpha (TNF-α) as stimulus. Applying inhibitors against the respiratory chain complexes, we identified mitochondria as primary source of ROS production. TNF-α altered mitochondrial integrity by mimicking a mild uncoupling effect in liver cells, as indicated by a 40% reduction in membrane potential and ATP depletion (35%). TNF-α-induced ROS production activated NF-κB 3.5-fold and subsequently enhanced migration up to 12.7-fold. This study identifies complex I and complex III of the mitochondrial respiratory chain as point of release of ROS upon TNF-α stimulation of liver cells, which enhances cell migration by activating NF-κB signalling.

Long-term exposure of human proximal tubule cells to hydroxycobalamin[c-lactam] as a possible model to study renal disease in methylmalonic acidurias.

Dysfunction of proximal tubules resulting in tubulointerstitial nephritis and chronic renal failure is a frequent long-term complication of methylmalonic acidurias. However, the underlying pathomechanisms have not yet been extensively studied owing to the lack of suitable in vitro and in vivo models. Application of hydroxycobalamin[c-lactam] has been shown to inhibit the metabolism of hydroxycobalamin and, thereby, to induce methylmalonic aciduria in rats, oligodendrocytes, and rat hepatocytes. Our study characterizes the biochemical and bioenergetic effects of long-term exposure of human proximal tubule cells to hydroxycobalamin[c-lactam], aiming to establish a novel in vitro model for the renal pathogenesis of methylmalonic acidurias. Incubation of human proximal tubule cells with hydroxycobalamin[c-lactam] and propionic acid resulted in a strong, time-dependent intra- and extracellular accumulation of methylmalonic acid. Bioenergetic studies of respiratory chain enzyme complexes revealed an increase of complex II-IV activity after 2 weeks and an increase of complex I and IV activity as well as a decrease of complex II and III activity after 3 weeks of incubation. In addition, human proximal tubule cells displayed reduced glutathione content after the exposure to hydroxycobalamin[c-lactam] and propionic acid.

Pathogenesis of CNS involvement in disorders of amino and organic acid metabolism.

Inherited disorders of amino and organic acid metabolism have a high cumulative frequency, and despite heterogeneous aetiology and varying clinical presentation, the manifestation of neurological disease is common. It has been demonstrated for some of these diseases that accumulating pathological metabolites are directly involved in the manifestation of neurological disease. Various pathomechanisms have been suggested in different in vitro and in vivo models including an impairment of brain energy metabolism, an imbalance of excitatory and inhibitory neurotransmission, altered transport across the blood-brain barrier and between glial cells and neurons, impairment of myelination and disturbed neuronal efflux of metabolic water. This review summarizes recent knowledge on pathomechanisms involved in phenylketonuria, glutaric aciduria type I, succinic semialdehyde dehydrogenase deficiency and aspartoacylase deficiency with examples, highlighting general as well as disease-specific concepts and their putative impact on treatment.

Neurodegeneration and chronic renal failure in methylmalonic aciduria--a pathophysiological approach.

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.

Biochemistry and bioenergetics of glutaryl-CoA dehydrogenase deficiency.

Glutaryl-CoA dehydrogenase (GCDH) is a central enzyme in the catabolic pathway of L-tryptophan, L-lysine, and L-hydroxylysine which catalyses the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA and CO2. Glutaryl-CoA dehydrogenase deficiency (GDD) is an autosomal recessive disease characterized by the accumulation of glutaric and 3-hydroxyglutaric acids in tissues and body fluids. Untreated patients commonly present with severe striatal degeneration during encephalopathic crises. Previous studies have highlighted primary excitotoxicity as a trigger of striatal degeneration. The aim of this PhD study was to investigate in detail tissue-specific bioenergetic and biochemical parameters of GDD in vitro, post mortem, and in Gcdh-/- mice. The major bioenergetic finding was uncompetitive inhibition of alpha-ketoglutarate dehydrogenase complex by glutaryl-CoA. It is suggested that a synergism of primary and secondary excitotoxic effects in concert with age-related physiological changes in the developing brain underlie acute and chronic neurodegenerative changes in GDD patients. The major biochemical findings were highly elevated cerebral concentrations of glutaric and 3-hydroxyglutaric acid despite low permeability of the blood-brain barrier for these dicarboxylic acids. It can be postulated that glutaric and 3-hydroxyglutaric acids are synthesized de novo and subsequently trapped in the brain. In this light, neurological disease in GDD is not 'transported' to the brain in analogy with phenylketonuria or hepatic encephalopathy as suggested previously but is more likely to be induced by the intrinsic biochemical properties of the cerebral tissue and the blood-brain barrier.

Enzymatic and metabolic evidence for a region specific mitochondrial dysfunction in brains of murine succinic semialdehyde dehydrogenase deficiency (Aldh5a1-/- mice).

Succinic semialdehyde dehydrogenase deficiency, a rare inherited defect of gamma-aminobutyrate (GABA) catabolism, presents with characteristic biochemical abnormalities in the central nervous system (CNS). These include elevated concentrations of GABA, gamma-hydroxybutyrate (GHB), succinic semialdehyde (SSA), 4,5-dihydroxyhexanoic acid (DHHA) and alanine as well as decreased concentrations of glutamine. GABA degradation is coupled to Krebs cycle function in mammalian CNS ("GABA shunt") through succinate and alpha-ketoglutarate. Accordingly, we hypothesized that disruption of Krebs cycle and respiratory chain function in the CNS is involved in the neuropathogenesis of this disease. For this purpose, we investigated cerebral activities of Krebs cycle and respiratory chain enzymes as well as the glutathione content in Aldh5a1(-/-) mice, a recently generated mouse model for this disease. In CNS tissue of Aldh5a1(-/-) mice, we found a significantly decreased glutathione content (hippocampus, cortex) and decreased activities of complexes I-IV (hippocampus) suggesting increased oxidative stress and mitochondrial dysfunction. However, specific activities of Krebs cycle and respiratory chain were not affected by GABA, GHB, SSA, or DHHA (up to 1 mmol/L). Although our results suggest hippocampal and cortical dysfunction in Aldh5a1(-/-) brain, we found no evidence that accumulating key metabolites of SSADH deficiency directly induce impairment of energy metabolism.

The aetiology of neurological complications of organic acidaemias--a role for the blood-brain barrier.

The blood-brain barrier (BBB) metabolically isolates the central nervous system (CNS) from the circulation and protects it against fluctuations of hydrophilic nutrients in plasma and from intoxication. Recent studies have shown that dicarboxylic acids (DCAs) are transported across the blood-brain barrier at very low rates. In organic acidaemias, neurological complications are common. We hypothesize that, as a result of the very limited efflux, in certain organic acidaemias there is pathological accumulation of DCAs (e.g. glutarate, 3-hydroxyglutarate, D-2- and L-2-hydroxyglutarate, methylmalonate) in the brain secondary to the metabolic block. At high concentrations some of these compounds may become neurotoxic. Treatment should be aimed at preventing the accumulation of these compounds using our understanding of the properties of the BBB.

Hydroxocobalamin: improved public health readiness for cyanide disasters.

The United States is under the constant threat of a mass casualty cyanide disaster from industrial accidents, hazardous material transportation incidents, and deliberate terrorist attacks. The current readiness for cyanide disaster by the emergency medical system in the United States is abysmal. We, as a nation, are simply not prepared for a significant cyanide-related event. The standard of care for cyanide intoxication is the cyanide antidote kit, which is based on the use of nitrites to induce methemoglobinemia. This kit is both expensive and ill suited for out-of-hospital use. It also has its own inherent toxicity that prevents rapid administration. Furthermore, our hospitals frequently fail to stock this life-saving antidote or decline to stock more than one. Hydroxocobalamin is well recognized as an efficacious, safe, and easily administered cyanide antidote. Because of its extremely low adverse effect profile, it is ideal for out-of-hospital use in suspected cyanide intoxication. To effectively prepare for a cyanide disaster, the United States must investigate, adopt, manufacture, and stockpile hydroxocobalamin to prevent needless morbidity and mortality.