Determination of Porphyrins in Bile Using High Performance Liquid Chromatography and Some Clinical Applications

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Bacteroides fragilis requires the ferrous-iron transporter FeoAB and the CobN-like proteins BtuS1 and BtuS2 for assimilation of iron released from heme

The intestinal commensal and opportunistic anaerobic pathogen Bacteroides fragilis has an essential requirement for both heme and free iron to support growth in extraintestinal infections. In the absence of free iron, B. fragilis can utilize heme as the sole source of iron. However, the mechanisms to remove iron from heme are not completely understood. In this study, we show that the inner membrane ferrous iron transporter ∆feoAB mutant strain is no longer able to grow with heme as the sole source of iron. Genetic complementation with the feoAB gene operon completely restored growth. Our data indicate that iron is removed from heme in the periplasmic space, and the released iron is transported by the FeoAB system. Interestingly, when B. fragilis utilizes iron from heme, it releases heme-derived porphyrins by a dechelatase activity which is upregulated under low iron conditions. This is supported by the findings showing that formation of heme-derived porphyrins in the ∆feoAB mutant and the parent strain increased 30-fold and fivefold (respectively) under low iron conditions compared to iron replete conditions. Moreover, the btuS1 btuS2 doublemutant strain (lacking the predicted periplasmic, membrane anchored CobN-like proteins) also showed growth defect with heme as the sole source of iron, suggesting that BtuS1 and BtuS2 are involved in heme-iron assimilation. Though the dechelatase mechanism remains uncharacterized, assays performed in bacterial crude extracts show that BtuS1 and BtuS2 affect the regulation of the dechelatase-specific activities in an iron-dependent manner. These findings suggest that the mechanism to extract iron from heme in Bacteroides requires a group of proteins, which spans the periplasmic space to make iron available for cellular functions

Evaluation and Clinical Application of the Enterotest® for the Determination of Human Biliary Porphyrin Composition

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Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport

Erythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of this hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process. Gel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates. Gel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic coproporphyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rat resulted in parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in impaired secretion into bile of protoporphyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Groningen Yellow rats with defective ATP-dependent hepatobiliary organic anion transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporphyrin I and III was affected to a much lesser extent. Our data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functioning coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretio

Homozygous acute intermittent porphyria in a 7-year-old boy with massive excretions of porphyrins and porphyrin precursors.

Item does not contain fulltextA 7-year-old boy demonstrating hepatosplenomegaly, mild anaemia, mild mental retardation, yellow-brown teeth and dark red urine had excessively elevated levels of urinary delta-aminolevulinic acid, porphobilinogen and uroporphyrin. Furthermore hepta-, hexa-, penta- and copro(I)porphyrins were highly increased in urine. This pattern of porphyrin precursor and metabolite excretion is characteristic of acute intermittent porphyria. The decreased copro(III)/copro(I+III) ratio, normally not found in acute intermittent porphyria, is discussed. The porphobilinogen deaminase activity in red cells was decreased to 2-4%. Mutation analysis revealed a novel homozygous L81P mutation in exon 6 of the porphobilinogen deaminase gene. The father and mother, shown to be gene carriers of the same mutation, are asymptomatic and have normal urinary porphyrin precursor and metabolite excretion