Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells

Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using the MnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300 μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300 μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria

Acute intermittent porphyria causes hepatic mitochondrial energetic failure in a mouse model

Acute intermittent porphyria (AIP), an inherited hepatic disorder, is due to a defect of hydroxymethylbilane synthase (HMBS), an enzyme involved in heme biosynthesis. AIP is characterized by recurrent, life-threatening attacks at least partly due to the increased hepatic production of 5-aminolaevulinic acid (ALA). Both the mitochondrial enzyme, ALA synthase (ALAS) 1, involved in the first step of heme biosynthesis, which is closely linked to mitochondrial bioenergetic pathways, and the promise of an ALAS1 siRNA hepatic therapy in humans, led us to investigate hepatic energetic metabolism in Hmbs KO mice treated with phenobarbital. The mitochondrial respiratory chain (RC) and the tricarboxylic acid (TCA) cycle were explored in the Hmbs−/− mouse model. RC and TCA cycle were significantly affected in comparison to controls in mice treated with phenobarbital with decreased activities of RC complexes I (−52%, **p < 0.01), II (−50%, **p < 0.01) and III (−55%, *p < 0.05), and decreased activity of α-ketoglutarate dehydrogenase (−64%, *p < 0.05), citrate synthase (−48%, **p < 0.01) and succinate dehydrogenase (−53%, *p < 0.05). Complex II-driven succinate respiration was also significantly affected. Most of these metabolic alterations were at least partially restored after the phenobarbital arrest and heme arginate administration. These results suggest a cataplerosis of the TCA cycle induced by phenobarbital, caused by the massive withdrawal of succinyl-CoA by ALAS induction, such that the TCA cycle is unable to supply the reduced cofactors to the RC. This profound and reversible impact of AIP on mitochondrial energetic metabolism offers new insights into the beneficial effect of heme, glucose and ALAS1 siRNA treatments by limiting the cataplerosis of TCA cycle

Mitochondrial energetic defects in muscle and brain of a Hmbs-/- mouse model of acute intermittent porphyria

Acute intermittent porphyria (AIP), an autosomal dominant metabolic disease (MIM #176000), is due to a deficiency of hydroxymethylbilane synthase (HMBS), which catalyzes the third step of the heme biosynthetic pathway. The clinical expression of the disease is mainly neurological, involving the autonomous, central and peripheral nervous systems. We explored mitochondrial oxidative phosphorylation (OXPHOS) in the brain and skeletal muscle of the Hmbs(-/-) mouse model first in the basal state (BS), and then after induction of the disease with phenobarbital and treatment with heme arginate (HA). The modification of the respiratory parameters, determined in mice in the BS, reflected a spontaneous metabolic energetic adaptation to HMBS deficiency. Phenobarbital induced a sharp alteration of the oxidative metabolism with a significant decrease of ATP production in skeletal muscle that was restored by treatment with HA. This OXPHOS defect was due to deficiencies in complexes I and II in the skeletal muscle whereas all four respiratory chain complexes were affected in the brain. To date, the pathogenesis of AIP has been mainly attributed to the neurotoxicity of aminolevulinic acid and heme deficiency. Our results show that mitochondrial energetic failure also plays an important role in the expression of the disease

A Plasma Metabolomic Signature Involving Purine Metabolism in Human Optic Atrophy 1 (OPA1)-Related Disorders

Purpose: Dominant optic atrophy (DOA; MIM [Mendelian Inheritance in Man] 165500), resulting in retinal ganglion cell degeneration, is mainly caused by mutations in the optic atrophy 1 (OPA1) gene, which encodes a dynamin guanosine triphosphate (GTP)ase involved in mitochondrial membrane processing. This work aimed at determining whether plasma from OPA1 pathogenic variant carriers displays a specific metabolic signature. Methods: We applied a nontargeted clinical metabolomics pipeline based on ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) allowing the exploration of 500 polar metabolites in plasma. We compared the plasma metabolic profiles of 25 patients with various OPA1 pathogenic variants and phenotypes to those of 20 healthy controls. Statistical analyses were performed using univariate and multivariate (principal component analysis [PCA], orthogonal partial least-squares discriminant analysis [OPLS-DA]) methods and a machine learning approach, the Biosigner algorithm. Results: A robust and relevant predictive model characterizing OPA1 individuals was obtained, based on a complex panel of metabolites with altered concentrations. An impairment of the purine metabolism, including significant differences in xanthine, hypoxanthine, and inosine concentrations, was at the foreground of this signature. In addition, the signature was characterized by differences in urocanate, choline, phosphocholine, glycerate, 1-oleoyl-rac-glycerol, rac-glycerol-1-myristate, aspartate, glutamate, and cystine concentrations. Conclusions: This first metabolic signature reported in the plasma of patient carrying OPA1 pathogenic variants highlights the unexpected involvement of purine metabolism in the pathophysiology of DOA

Validation of plasma amino acid profile using UHPLC-mass spectrometer (QDa) as a screening method in a metabolic disorders reference centre: Performance and accreditation concerns

International audienceINTRODUCTION: Amino acid (AA) analysis in plasma is essential for diagnosis and monitoring of inborn errors of metabolism (IEM). The efficacy of patient management is governed by the rapidity of AA profile availability, along with the robustness of the method. French quality guidelines and progress made in analytical techniques have led biologists to develop AA profile exploration via mass spectrometry (MS). OBJECTIVES: The aim of this study was to validate an analytical method with a single quadrupole mass spectrometer (MS) and to suggest reference values in regard to French quality and IEM society recommendations. DESIGN AND METHODS: Plasma samples from patients were deproteinised and derivatised with AccqTag™ reagent. Analysis was performed by reverse-phase chromatography coupled to QDA detector. We evaluated accuracy, intra-days and inter-days precision and limit of quantification by the β-expectation tolerance interval method for 27 AA. Method comparison was performed with the standard method (ion exchange chromatography, IEC) on Jeol Aminotac® and to tandem MS. Reference values were established on AA concentrations of the cohort of patients who had no IEM. RESULTS: Our method allowed the separations of almost all amino acids with a total run time of 12 min. Separation of isoleucine and alloisoleucine was incomplete (R = 0.55) but without impact on biological interpretation. Precision, accuracy and quantification were satisfactory (intra-days coefficient of variation (CV) was <5%, inter-days precision CV <10% and accuracy <15%). The limits of quantification were validated between 1 and 900 µmol/L. Results were comparable between the new method and IEC. CONCLUSION: Ultimately, we validated a rapid method on plasma for quantifying 27 amino acids that can be used in routine practice, according to French quality laboratories and SFEIM (French Society of Inborn Error of Metabolism) recommendations. Furthermore, estimated reference values were similar to those found in published studies focusing on other methods. Despite a lower specificity compared to tandem MS, the simplicity and rapidity of our method are the main advantage of this technique and place it as a major tool in IEM diagnosis and management

Pituitary stalk interruption syndrome: a rare and severe cause of pituitary deficiency Laboratory diagnosis of a newborn case

We report the case of a newborn with neonatal hypotonia associated to a micropenis and a bilateral cryptorchidia. The discovery of severe hypoglycemia at 0.22 mmol/L led to further biological investigations that revealed sharply decreased levels of several hypophyseal hormones. Altered corticotropic, somatotropic, thyreotropic, and gonadotropic axes finally suggested congenital hypophyseal insufficiency. This diagnostic was confirmed by a brain MRI (magnectic resonance imaging), which revealed a total interruption of the pituitary stalk. Immediate substitutive hormonal treatment allowed a clinical improvement of the condition and limited the risk of further episodes of hypoglycemia. The pituitary stalk interruption syndrome (PSIS), a very rare congenital disorder, has an estimated incidence of about 1:200.000. This developmental anomaly of the hypophysis calls for urgent diagnosis since prognosis depends on the rapid implementation of substitutive hormonal therapy. The hormonal deficit in the newborn affected by PSIS is often of a multiple nature with a constant somatotropic deficit, thus requiring the exploration of the different antehypophyseal axes. Despite the fact that PSIS is a rare disorder, it should always be kept in the differential diagnosis of newborn presenting with hypoglycemia. Since the interpretation of hormonal assays is particularly delicate at birth, close clinico-biological cooperation is essential for rapid diagnosis of PSIS and appropriate adaptation of the short- and long-term therapeutic management of the newborn

Systemic messenger RNA as an etiological treatment for acute intermittent porphyria

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP