High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

Bonkovsky, Herbert L.; Wood, Sheryl G.; Howell, Scott K.; Sinclair, Peter R.; Lincoln, Beth; Healey, John F.; Sinclair, Jacqueline F.

doi:10.1016/0003-2697(86)90224-1

Cited by 66 publications

(34 citation statements)

References 31 publications

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“…28 The product coproporphyrinogen was oxidized to coproporphyrin and quantified by high-performance liquid chromatography (HPLC). 29 With this assay, the mean (range) activity for normals is 3.5 nmol coproporphyrinogen I · h Ϫ1 · mg protein Ϫ1 (2.7-4.5), and for familial (type II) PCT, it was 1.5 nmol coproporphyrinogen I · h Ϫ1 · mg protein Ϫ1 (0.9-2.4).…”

Section: Methodsmentioning

confidence: 92%

Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in north america†

et al. 1998

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In some, but not all countries, porphyria cutanea tarda (PCT) has been associated with chronic infection with the hepatitis C virus (HCV). Recently, PCT has also been associated with mutations in the HFE gene that are associated with HLA-linked hereditary hemochromatosis. Until now, few studies of these associations have been reported from North America. The aims of this study were: 1) to assess the prevalence of HCV infection and HFE mutations in North American patients with PCT; 2) to compare demographic and laboratory features between those who are HCV-positive and HCV-negative; and 3) to study urinary porphyrin excretions in American HCV-positive patients without clinically manifest PCT. Clinical and laboratory data, including tests for HCV and urinary porphyrins, were collected from 70 unselected patients with typical PCT. Urinary porphyrins were also measured in 110 non-PCT patients with chronic hepatitis C. Mutational analyses of the HFE gene were performed in 26 PCT patients. Thirtynine of 70 (56%) of the PCT patients had evidence of HCV infection. Thirty-two of 39 PCT patients with HCV were men, all of whom used alcohol. In contrast, 22 of 31 PCT patients without HCV infection were women, 12 of whom had taken estrogens. The HCV-positive group was more likely to have used illicit intravenous drugs (45% vs. 0%; P ‫؍‬ 0.01), to have had several (G4) sex partners (48% vs. 13%; P ‫؍‬ 0.005), and less likely to have no known risk factors for HCV infection (33% vs. 78%; P ‫؍‬ 0.004). Total urinary porphyrin excretion was the same in the two groups, but those with HCV infection had a significantly lower percentage of uroporphyrin and higher percentages of hepta-and hexa-carboxy porphyrins in urine. Sixteen of 110 (15%) HCV-positive subjects without PCT had increased urinary porphyrins, but, unlike PCT, these were mainly coproporphyrin. Forty-two percent of PCT patients carried the C282Y mutation of HFE (15% homozygous), and another 31% carried the H63D mutation (8% homozygous). Thus, 73% of PCT patients had one of these mutations. The prevalence of HCV infection (56%) and mutations in the HFE gene (73%) are high among North American patients with PCT. Alcohol and estrogen use are important additional risk factors. All PCT patients should be tested for HCV infection and for HFE gene mutations. Although HCV infection is a trigger for PCT, preclinical PCT is rare in chronic HCV hepatitis C in the United States. (HEPATOLOGY 1998;27:1661-1669.)

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Section: Methodsmentioning

confidence: 92%

Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in north america†

et al. 1998

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“…After centrifugation to remove the precipitated protein, the concentrations of coproporphyrin, uroporphyrin, and protoporphyrin IX present in the plastids or the supernatant were quantitated in PCA:methanol on a Perkin-Elmer 650 10s spectrofluorometer by the method of Grandchamp et al (1980). The identity of the porphyrins in several assays was confirmed by HPLC (Bonkovsky et al, 1986) and by TLC of the free porphyrins (Henderson, 1989) to verify the spectrofluorometric assay. These fluorescence assays determine porphyrins and not porphyrinogens; most porpliyrinogens, however, are converted to their corresponding porphyrins after several hours in acidic solvents under aerobic conditions.…”

Section: Measurement Of Porphyrin-synthesizing Activitymentioning

confidence: 99%

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

Jacobs

1993

Plant Physiol.

137

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We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presente and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen I X to protoporphyrin IX.In the absence of herbicide, about 50% of the protoporphyrin I X formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period. In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid. When the incubation was carried out in the-presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium. To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escbericbia coli membranes.Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX. Protoporphyrin I X and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction. These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm. These results help explain the extraplastidic accumulation of protoporphyrin I X in plants treated with photobleaching herbicides. In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis.There has been increased interest in the mechanism of plant Chl and heme synthesis due to recent studies showing that the light-dependent action of photobleaching diphenylether herbicides involves the accumulation of protoporphyrin IX, an intermediate in the heme and Chl synthetic pathways. This photoactive porphyrin can generate membrane-damag-

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“…Treatment sets of 50 cotyledons were extracted; the porphyrins were evaporated to dryness from ether, then taken up in methanol and chromatographed on a reversed-phase C18 column according to Bonkovsky et al (3). Major peaks absorbing at 400 nm are designated by retention time in minutes.…”

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confidence: 99%

“…Under green safelights, excised cotyledons were placed on media treated with 300 ,M AFM and were dark-incubated for 6 h; 5 mM ALA was then added to all treatments, which were held for an additional 16 h in darkness. Treatment sets of 50 cotyledons were extracted; the porphyrins were evaporated to dryness from ether, then taken up in methanol and chromatographed on a reversed-phase C18 column as according to Bonkovsky et al (3). Major peaks absorbing at 400 nm are designated by retention time in minutes.…”

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Accumulation of Photodynamic Tetrapyrroles Induced by Acifluorfen-Methyl

Witkowski

Halling²

1988

Plant Physiol.

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Treatment with acifluorfen-methyl (AFM), methyl 542-chloro-4-Itrifluoromethyll phenoxy)-2-nitrobenzoate, inhibited protochlorophyllide synthesis in dark-held, 5-amino levulinic acid-fed, excised cotyledons of cucumber (Cucumis sativus L.). Protochlorophyllide and protoporphyrin IX levels in AFM-treated cotyledons were inversely related and dependent on AFM concentration; as the herbicide dose increased, protoporphyrin IX levels also increased with a concomitant loss of protochlorophyllide. Significant protoporphyrin IX accumulation was induced by concentrations of AFM from the linear region of the membrane disruption dose response curve. The patterm of precursor accumulation seen in HPLC chromatograms from extracts of AFM-treated tissue indicate that Mg insertion into the tetrapyrrole ring is inhibited, suggesting interference with Mg-chelatase. An inhibitor of 5-amino levulinic acid synthesis, gabaculine (3-amino-2,3-dihydrobenzoic acid), completely blocked the membrane disruption activity of AFM in illuminated cotyledons. Protoporphyrin IX accumulating in AFM-treated tissues may serve as the primary photosensitizer for initiating lipid peroxidation.As a consequence of the rapid induction of lipid peroxidation, illumination ofplant tissues treated with p-nitro DPEI herbicides results in the disruption ofa wide range ofbiological constituents and functions (18,26). This broadly destructive activity by the DPE herbicides has made it difficult to monitor specific interactions between these compounds and light-dependent in vivo physiological processes. These effects lead to one of the most readily visible manifestations of the treatment of whole plants with DPE herbicides: the bleaching of the foliar tissue as a consequence of pigment photooxidation.In an earlier study (15) greening was induced in AFM-treated cucumber cotyledons without triggering a loss in plasmalemma membrane integrity under a regime oflow intensity, intermittent illumination. Yet, even under these conditions, we observed significant reductions in the levels of Chl accumulated in AFMtreated tissue. These results suggested the possibility that, in addition to the pigment bleaching in DPE-treated plant tissue which occurs under continuous illumination, AFM might also directly inhibit pigment biosynthesis in a manner not related to photooxidation.We have since found further evidence that the DPE herbicides directly inhibit Chl synthesis prior to the formation of Pchl(ide).' Abbreviations: DPE, diphenyl ether; AFM, acifluorfen-methyl; ALA, 6-amino levulinic acid; Pchl(ide), a mixture of Pchlide and Pchlide ester; proto IX, protoporphyrin IX; Mg-proto IX, Mg-protoporphyrin IX; Mgproto IX Me, Mg-protoporphyrin IX monomethyl ester; LA, levulinic acid: LSD (0.05), least significant difference at the 5% probability level.

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High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

Cited by 66 publications

References 31 publications

Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in north america†

Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in north america†

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

Accumulation of Photodynamic Tetrapyrroles Induced by Acifluorfen-Methyl

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