Measurement of <scp>ALA</scp> Synthase Activity

Sinclair, Peter R.; Gorman, Nadia; Cornell, Neal W.

doi:10.1002/0471140856.tx0802s00

Cited by 3 publications

(2 citation statements)

References 17 publications

(37 reference statements)

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“…The assay was conducted using a previous protocol with slight modification. 15 Briefly, 50 μg of mitochondria was incubated in 100 μl glycine buffer (35 mM Tris-HCl, pH 7.4, 30 mM Na 2 HPO 4 , 8 mM MgCl 2 , 0.2 mM pyridoxal phosphate, 25 μM succinylacetone, 5 mM EDTA and 150 mM glycine) for 1 h at 37 °C. The product of ALAS was analyzed using UPLC-QTOFMS as described previously 16 …”

Section: Methodsmentioning

confidence: 99%

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver

Sachar

Liu

et al. 2016

Chem. Res. Toxicol.

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Isoniazid (INH) can cause hepatotoxicity. In addition, INH is contraindicated in patients suffering from porphyrias. Our metabolomic analysis revealed that chronic treatment with INH in mice causes a hepatic accumulation of protoporphyrin IX (PPIX). PPIX is an intermediate in the heme biosynthesis pathway and it is also known as a hepatotoxin. We further found that INH induces delta-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. We also found that INH downregulates ferrochelatase (FECH), the enzyme that converts PPIX to heme. In summary, this study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation. This study also highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.

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Section: Methodsmentioning

confidence: 99%

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver

Sachar

Liu

et al. 2016

Chem. Res. Toxicol.

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“…16 ALAS activity in the freshly made mitochondrial fraction was determined as described. 29 The presence of CYP1A1, CYP1A2, CYP2E1, and CYP3A proteins in liver homogenates was determined by immunoblotting, following the electrophoretic separation of proteins in liver homogenates as described. 16 Immunoreactive proteins were quantitated by scanning digitized blot images with One-Dscan software (Scanalytics Inc., Fairfax, VA).…”

Section: Methodsmentioning

confidence: 99%

Uroporphyria caused by ethanol in Hfe (−/−) mice as a model for porphyria cutanea tarda

et al. 2003

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Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(؊/؊) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(؊/؊) but not wild-type mice. The increases in nonheme iron in Hfe(؊/؊) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(؊/؊) mice seem to be an excellent model for studies of alcohol-mediated PCT. (HEPATOLOGY 2003;37:351-358.)

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Regulation of human liver δ-aminolevulinic acid synthase by bile acids

et al. 2007

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Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acidactivated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5 flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at ؊13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/ retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias. (HEPATOLOGY 2007;46:1960-1970

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Measurement of ALA Synthase Activity

Cited by 3 publications

References 17 publications

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver

Uroporphyria caused by ethanol in Hfe (−/−) mice as a model for porphyria cutanea tarda

Regulation of human liver δ-aminolevulinic acid synthase by bile acids

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