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.

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.

Assessment of new hyaluronic acid assays and their impact on FibroMeter scores.

BACKGROUND: We compared three hyaluronic acid (HA) assays and analyzed the impact of their variations on FibroMeter scores. METHODS: In a test group of 165 patients, HA levels were assessed with the commonly used ELISA assay from Corgenix, a new ELISA assay from Teco and an immunoturbidimetry assay from Wako, this latter tested across three different instruments. Five different FibroMeter scores were calculated. RESULTS: Correlation across the three assays (r(s) between 0.969 and 0.995) was very good. Means of differences (d) were lower when the immunoturbidimetry assay was compared on different instruments: d between -3.4 and 2.0 µg/L. However, a higher value for HA measurement was observed with Corgenix assay, compared to the other two assays (Teco and Wako): d between 27.1 and 36.4 µg/L. The assessment also demonstrated that HA variations had very little impact on FibroMeter scores: 0.0117 for virus and 0.0416 for alcoholic fibrosis scores, and between 0.58 and 1.71 for the area of fibrosis (expressed in percentage). CONCLUSIONS: The two new assays found lower values of HA, as compared to the Corgenix assay. However, these differences had very little impact on FibroMeter scores and had no impact on clinical evaluation of liver fibrosis.

Comparison of blood tests for liver fibrosis specific or not to NAFLD.

BACKGROUND/AIMS: To compare blood tests of liver fibrosis specific for NAFLD: the FibroMeter NAFLD and the NAFLD fibrosis score (NFSA) with a non-specific test, APRI. METHODS: Two hundred and thirty-five NAFLD patients with liver Metavir staging and blood markers from two independent centres were randomly assigned to a test (n=121) or a validation population (n=114). RESULTS: The highest accuracy--91%--for significant fibrosis was obtained with the FibroMeter whose (i) AUROC (0.943) was significantly higher than those of NFSA (0.884, p=0.008) and APRI (0.866, p<10(-3); p=0.309 vs NFSA) in the whole population, and (ii) misclassification rate (9%) was significantly lower than those of NFSA (14%, p=0.04) and APRI (16%, p=0.002) and did not vary according to centre (14 vs 7%, p=0.07), unlike those of NFSA (25 vs 9%, p=0.001) and APRI (29 vs 11%, p<10(-3)). By using thresholds of 90% predictive values, liver biopsy could have been avoided in most patients: FibroMeter: 97.4% vs NFSA: 86.8% (p<10(-3)) and APRI: 80.0% (p<10(-3)). A new classification provided three reliable diagnosis intervals: F0/1, F0/1/2, F2/3/4 with 91.4% accuracy for FibroMeter, avoiding biopsy in all patients. CONCLUSIONS: FibroMeter NAFLD had high performance and provided reliable diagnosis for significant fibrosis, significantly outperforming NFSA and APRI.