The spectrophotometric determination of uroporphyrinogen i synthetase activity

Grandchamp, Bernard; Phung, N; Grelier, Mireille; Nordmann, Y

doi:10.1016/0009-8981(76)90011-5

Cited by 38 publications

(13 citation statements)

References 8 publications

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“…The activity of PBGD is measured by the absorbance of uroporphyrin, formed after light-induced oxidation of uroporphyrinogen, the immediate product of the enzymatic deamination reaction according to Grandchamp et al (1976). Briefly, cultured cells were removed with trypsin at selected times during incubations as described above, then transferred to 15-ml conical tubes and centrifuged at 1000 g for 5 min.…”

Section: Measurement Of Pbgd Activity In Cultured R3230ac Cellsmentioning

confidence: 99%

δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

Havens

Nguyen

et al. 1999

Br J Cancer

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Summary Recently, considerable interest has been given to photodynamic therapy of cancer using δ-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after δ-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with δ-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM δ-aminolaevulinic acid for 27 h accumulated 6.07 × 10 -16 mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm -2 irradiation after a 3-h incubation with δ-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 × 10 -16 mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with δ-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the δ-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.

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Section: Measurement Of Pbgd Activity In Cultured R3230ac Cellsmentioning

confidence: 99%

δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

Havens

Nguyen

et al. 1999

Br J Cancer

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“…The procedure is essentially described by Grandchamp et al (1976). Briefly, tissues are homogenized (1:5 w/v) in 0.05 M Tris-HCl (pH 7.4), centrifuged at lOOOg for 15 min and portions of the supematant containing 2 mg of protein are incubated for 30 min at 45°C in the dark with 1.0 ml of porphobilinogen at concentrations ranging from 0 to 500 JM.…”

Section: Detection Of Porphyrin Fluorescence In Tissue Homogenatesmentioning

confidence: 99%

A regulatory role for porphobilinogen deaminase (PBGD) in δ-aminolaevulinic acid (δ-ALA)-induced photosensitization?

Gibson

Cupriks²,

Havens³

et al. 1998

Br J Cancer

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Summary As an initial approach to optimize 8-aminolaevulinic acid (6-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: 8-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time-and dose-related increase in tumours after intravenous administration of 300 mg kg-1 6-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mm 6-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after 6-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a ratelimiting determinant for the efficacy of 6-ALA-induced photosensitization when used in photodynamic therapy.Keywords: 6-aminolaevulinic acid: photosensitization; porphobilinogen deaminase: haem biosynthesis; porphyrin fluorescence Haem is an essential prosthetic group in many critical cellular proteins such as haemoglobin, cytochrome P450 and cyclooxygenase (Abraham, 1991). Eight enzymes are involved in the biosynthesis of haem, a process that occurs in two subcellular compartments: the mitochondria and the cytosol. The first enzyme in the haem pathway, mitochondrial 8-aminolaevulinic acid synthase (8-ALA-S), forms 6-ALA from glycine and succinyl CoA and is a target for the regulation of haem biosynthesis. Feedback inhibition of 8-ALA-S occurs when intracellular haem is present in excess (Ade, 1990).The last step in the haem biosynthetic pathway, the insertion of iron into PPIX to form haem, is catalysed by the mitochondrial enzyme ferrochelatase (FC). In unperturbed systems, FC is regulated by the availability of iron and/or PPIX. Two of the metabolic events that occur between 8-ALA-S and FC are catalysed by the cytosolic enzymes 8-ALA dehydratase (8-ALA-D) and porphobilinogen deaminase (PBGD). The dehydratase enzyme catalyses the condensation of two 8-ALA molecules to form porphobilinogen (PBG). It is the first enzyme that will metabolize the administered 8-ALA. The next enzyme in haem biosynthesis, PBGD, forms the tetrapyrole ring from four porphobilinogen molecules (Abraham, 1991).By providing the 8-ALA-S product, 8-ALA, the initial feedback step has been circumvented and this approach has been exploited for use in photodynamic therapy (PDT) of cancer (Kennedy and Pottier, 1992;Grant et al, 1993; Cairnduff et al, 1994;Regula et al, 1995). Traditional PDT regimens consist of the systemic adminis- Accepted 13 June 1997 Correspondence to: R Hilf tration of a photosensitizer followed by an appropriate interval to allow for its maximal accumulation in ma...

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“…Erythrocyte HMBS activity was evaluated by spectrofluorimetric or spectrophotometric assays (Grandchamp et al, 1976) using 5 mL of blood. Reference range values were calculated using mean ± standard deviation of the measured HMSB activity.…”

Section: Hmbs Activitymentioning

confidence: 99%

“…Usually the diagnosis is based upon levels of urine porphyrins and their precursors during the crisis. Red cell HMBS activity assaying is performed by spectrofluorimetry or by spectrophotometry Grandchamp et al, 1976). However, levels of enzyme activity are insufficient to confirm that a decreased activity may identify a carrier, as enzyme levels may overlap between normal controls and carriers (Bottomley et al, 1981;Bonaitie-Pellié et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Hydroxymethylbilane Synthase Gene Mutations and Polymorphisms in Brazilian Families with Acute Intermittent Porphyria

Gonzaga

Amorim

Fonseca

et al. 2015

Annals of Human Genetics

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SummaryAcute intermittent porphyria (AIP), an autosomal dominant disorder, is caused by a deficiency of hydroxymethylbilane synthase (HMBS). In the present study, we sought to establish a correlation between HMBS activity with the presence of mutations and polymorphisms. Enzyme activity was measured in red blood cells of four Brazilian unrelated AIP families (n = 124) and in blood donors (n = 80). The HMBS mutations in AIP family members were studied by PCR-SSCP followed by direct sequencing. Six intragenic SNPs (1345 G>A, 1500 T>C, 2377 C>A, 2478 A>G, 3581 A>G, and 7064 C>A) were determined by PCR-RFLP. Abnormal SSCP patterns in exons 7, 9, 12, and 15 were observed. DNA sequencing analysis revealed one nonsense mutation, R149X, two missense mutations, G111R and L338P, and one deletion, CT 730-731. All mutation carriers had lower enzyme activity. All polymorphisms, except 2377 C>A and 7064 C>A, showed no significant differences compared with previous reports. Mutation screening allowed the detection of the missense mutation, L338P, and the 730_731delCT deletion, two as yet unreported mutations in Brazilian AIP patients. Our findings also showed a high frequency of 2478 A>G and 3581 A>G polymorphism combinations suggesting that these polymorphisms contributed to enzymatic activity reduction in our study population.

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The spectrophotometric determination of uroporphyrinogen i synthetase activity

Cited by 38 publications

References 8 publications

δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

δ-Aminolaevulinic acid-induced photodynamic therapy inhibits protoporphyrin IX biosynthesis and reduces subsequent treatment efficacy in vitro

A regulatory role for porphobilinogen deaminase (PBGD) in δ-aminolaevulinic acid (δ-ALA)-induced photosensitization?

Hydroxymethylbilane Synthase Gene Mutations and Polymorphisms in Brazilian Families with Acute Intermittent Porphyria

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