Coproporphyrin III Research Articles

Significant increases in detection capability for assessment of organic anion transporting polypeptide-mediated drug-drug interaction in cynomolgus monkeys by modifying pretreatment of Coproporphyrin I and III, and glycochenodeoxycholate-3-sulfate in plasma

BackgroundCoproporphyrin I (CP–I), Coproporphyrin III (CP-III), and glycochenodeoxycholate-3-sulfate (GCDCA-S) act as endogenous substrates of Organic Anion Transporting Polypeptide (OATP) 1B and have been considered for application in OATP1B-mediated drug‒drug interaction (DDI) risk assessments. Prior assays of the endogenous OATP substrates might exhibit reduced DDI detection capability and possibly overlook low DDI risk. We pioneered a simultaneous assay of the three substrates in monkey plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) and applied it to monkey studies to identify lower DDI risk. ResultsThe methodology development indicated that precursors of CP-I/III were oxidized to form CP-I/III, diminishing the detection capability in DDI risk assessments. A precursor eliminated analytical (PEA) method was developed to eliminate the precursors through solid-phase extraction. This method aimed to prevent the oxidation of CP-I/III precursors by incorporating edaravone. For comparison, a precursor oxidized analytical (POA) method was also developed, wherein the precursors of CP-I/III were fully oxidized to CP-I/III. The PEA method achieved high sensitivity for CP-I/III and GCDCA-S, with lower quantification limits of 0.01 ng mL−1 and 0.5 ng mL−1, respectively. Both methods ensured that the validation parameters met the acceptance criteria. The two methods were applied to a monkey study, with CP-I/III showcasing notably enhanced DDI detection capabilities through the novel PEA method in comparison to the POA method. SignificanceThis study's methodology has future implications for OATP-mediated DDI risk assessment using endogenous substrates. The novel PEA method can identify lower OATP-mediated DDI risks for drugs that the current methods cannot detect. Our method is likely applicable in clinical settings, and its utility should be assessed in clinical trials.

High-yield porphyrin production through metabolic engineering and biocatalysis.

Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L-1 by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L-1 and 21.3 g L-1, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.

An Isolated Perfused Rat Liver Model: Simultaneous LC-MS Quantification of Pitavastatin, Coproporphyrin I, and Coproporphyrin III Levels in the Rat Liver and Bile.

The isolated perfused rat liver (IPRL) model provides a mechanistic understanding of the organic-anion-transporting polypeptide (OATP/Oatp)-mediated pharmacokinetics in the preclinical evaluation, which often requires the use of control substrates (i.e., pitavastatin) and monitoring endogenous biomarkers (coproporphyrin I and III). This study aimed to develop and validate an LC-MS method allowing the simultaneous quantification of pitavastatin, coproporphyrin I (CPI), and coproporphyrin III (CPIII) in rat liver perfusion matrices (perfusate, liver homogenate, bile). The analysis was performed on a C18 column at 60 °C with 20 μL of sample injection. The mobile phases consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid with a gradient flow of 0.5 mL/min. The assay was validated according to the ICH M10 Bioanalytical Method Validation Guideline (2022) for selectivity, calibration curve and range, matrix effect, carryover, accuracy, precision, and reinjection reproducibility. The method allowing the simultaneous quantification of pitavastatin, CPI, and CPIII was selective without having carryover and matrix effects. The linear calibration curves were obtained within various calibration ranges for three analytes in different matrices. Accuracy and precision values fulfilled the required limits. After 60 min perfusion with pitavastatin (1 μM), the cumulative amounts of pitavastatin in the liver and bile were 5.770 ± 1.504 and 0.852 ± 0.430 nmol/g liver, respectively. CPIII was a more dominant marker than CPI in both liver (0.028 ± 0.017 vs 0.013 ± 0.008 nmol/g liver) and bile (0.016 ± 0.011 vs 0.009 ± 0.007 nmol/g liver). The novel and validated bioanalytical method can be applied in further IPRL preparations investigating Oatp-mediated pharmacokinetics and DDIs.

Influence of vitamins and food on the fluorescence spectrum of human urine.

Fluorescence spectroscopy of human urine is a method with the potential to gain importance as a diagnostic tool in the medical field, e.g.,for measuring Coproporphyrin III (CPIII) as an indicator of cancer and acute types of porphyria. Food can change human urine's color, which could influence the urine fluorescence spectrum and the detection of CPIII in urine. To determine if there is a noticeable influence on the urine fluorescence spectrum or on the detection of CPIII in urine, 16vitamin supplements, and three food items were tested. Such investigation may also prevent false interpretation of measured data. Urine samples were collected before and after (overnight, ca. 8 h) intake of each test substance. Samples were investigated by fluorescence spectrum analysis. At excitation wavelengths from 300to 500 nm and emission wavelengths from 400to 700 nm excitation-emission-matrices were measured. Data obtained from urine before intake were compared to the data from overnight urine. Furthermore, the investigation of any interference with the CPIII concentration was performed at an excitation wavelength of 407 ± 3 nm and emission wavelengths of 490-800 nm. Only vitamin B2, but none of the other tested substances, showed noticeable influence on the urine fluorescence spectrum. None of the tested substances showed noticeable interference with the recovery rate of CPIII. The correct interpretation of measured data by fluorescence spectroscopy is possible with the exception if vitamin B2 supplementation was performed; thus, the consumption of vitamin B2 supplements before fluorescence testing of the patient's urine should be avoided and/or must be requested. CPIII concentrations could reliably be measured in all cases.

Unraveling the blue shift in porphyrin fluorescence in glioma: The 620 nm peak and its potential significance in tumor biology.

In glioma surgery, the low-density infiltration zone of tumors is difficult to detect by any means. While, for instance, 5-aminolevulinic acid (5-ALA)-induced fluorescence is a well-established surgical procedure for maximizing resection of malignant gliomas, a cell density in tumor tissue of 20-30% is needed to observe visual fluorescence. Hyperspectral imaging is a powerful technique for the optical characterization of brain tissue, which accommodates the complex spectral properties of gliomas. Thereby, knowledge about the signal source is essential to generate specific separation (unmixing) procedures for the different spectral characteristics of analytes and estimate compound abundances. It was stated that protoporphyrin IX (PpIX) fluorescence consists mainly of emission peaks at 634 nm (PpIX634) and 620 nm (PpIX620). However, other members of the substance group of porphyrins fluoresce similarly to PpIX due to their common tetrapyrrole core structure. While the PpIX634 signal has reliably been assigned to PpIX, it has not yet been analyzed if PpIX620 might result from a different porphyrin rather than being a second photo state of PpIX. We thus reviewed more than 200,000 spectra from various tumors measured in almost 600 biopsies of 130 patients. Insufficient consideration of autofluorescence led to artificial inflation of the PpIX620 peak in the past. Recently, five basis spectra (PpIX634, PpIX620, flavin, lipofuscin, and NADH) were described and incorporated into the analysis algorithm, which allowed more accurate unmixing of spectral abundances. We used the improved algorithm to investigate the PpIX620 signal more precisely and investigated coproporphyrin III (CpIII) fluorescence phantoms for spectral unmixing. Our findings show that the PpIX634 peak was the primary source of the 5-ALA-induced fluorescence. CpIII had a similar spectral characteristic to PpIX620. The supplementation of 5-ALA may trigger the increased production of porphyrins other than PpIX within the heme biosynthesis pathway, including that of CpIII. It is essential to correctly separate autofluorescence from the main PpIX634 peak to analyze the fluorescence signal. This article highlights the need for a comprehensive understanding of the spectral complexity in gliomas and suggests less significance of the 620 nm fluorescence peak for PpIX analysis and visualization.

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible “bulge” loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Yin and Yang of skin microbiota in "swimmer acne".

Cutibacterium acnes (C.acnes) is an organism implicated in the pathogenesis of acne. Despite regular immersion in antimicrobial chlorine, adolescent swimmers suffer from acne and tend to be resistant to standard therapies. Given the presence of Pseudomonas within swimming facilities, we hypothesized that "swimmer acne" is potentially driven by a different microbial mechanism. In this study, we aimed to examine the microbial dynamics of C.acnes and Pseudomonadaceae, a family of gram-negative bacteria (includes Pseudomonas aeruginosa), in swimmers and its potential contribution to the pathogenesis of acne in this population. Using fluorescence photography that measures the Coproporphyrin III (CPIII), we quantitated an absolute abundance of C.acnes present on the face of each participant pre- and post-swimming. In addition, 16S rRNA gene sequencing was utilized to assess relative abundance of the skin microbiota on each participant pre- and post-swimming. 16swimmers (8girls and 8 boys) completed the study. Seven had acne on the face. The CPIII fluorescence levels decreased for all swimmers after 1h of swimming (p-value <0.001). In contrast, the relative abundance of C.acnes remained unchanged, while that of Pseudomonadaceae increased after swimming (p-value =0.027). Comparing the relative abundances of Pseudomonadaceae before swimming, there was a significant increase in variance from the mean in acne group as compared to no acne group (p-value <0.001). Taken together, we conclude that the skin dysbiosis resulting from repeated decolonization and colonization of C.acnes and Pseudomonadaceae, respectively, can potentially be associated with the pathogenesis of acne in swimmers.

Endoscopic filter fluorometer for detection of accumulation of Protoporphyrin IX to improve photodynamic diagnostic (PDD)

Abstract Photodynamic diagnostic (PDD) is an optical enhancement option for the endoscope to support the detection of cancer, for example in the bladder. In real application PDD efficiency suffers due to the complex accumulation of the photosensitizing drug inside the tumor and the associated processes of heme syntheses to create the fluorescent components needed. To optimize the diagnostic outcome of PDD it would be helpful to predict the optimal time for diagnosis based on measurable precursors. In a previous cell study, we proposed a new filter fluorometer to image the accumulation of the precursors Coproporphyrin III (CP-III) and Uroporphyrin III (UP-III) that metabolize to Protoporphyrin IX (PP-IX) later. This accumulation process can be used to predict the optimal time slot for diagnostic imaging. Therefore, a new filter system was designed to distinguish between CP-III and PP-IX. In this work we tested this filter system in combination with a standard PDD endoscopic imaging system. Goal of this study was to prove the technical feasibility in a non-patient setup to prepare a later clinical study.

The importance of porphyrins in blue light suppression of Streptococcus agalactiae

It is well documented that blue light absorption by bacterial chromophores triggers downstream production of reactive oxygen species (ROS), which in turn results in bacterial cell death. To elucidate the importance of chromophores in the bactericidal effect of blue light, and to determine whether blue light absorption per se or the presence of porphyrins known to engender ROS is crucial in blue light treatment, we studied the effect of 450 nm pulsed light on Streptococcus agalactiae, also known as Group B Streptococcus (GBS) strain COH1. GBS does not synthesize porphyrins but has a blue light-absorbing chromophore, granadaene. We irradiated planktonic cultures of GBS with or without exogenous chromophore supplementation using either protoporphyrin IX (PPIX), coproporphyrin III (CPIII), Nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide (FAD), or Flavin mononucleotide (FMN). Quantification of surviving bacterial colonies, presented as percent survival and CFU/mL (log10), showed that (1) 450 nm blue light does not suppress the growth of GBS, even though its endogenous chromophore, granadaene, absorbs light in the 450 nm spectrum. (2) The addition of either of the two exogenous porphyrins, PPIX or CPIII, significantly suppressed GBS, indicating the importance of porphyrins in the antimicrobial action of blue light. (3) Adding exogenous FMN or FAD, two known absorbers of 450 nm light, minimally potentiated the bactericidal effect of blue light, again confirming that mere absorption of blue light by chromophores does not necessarily result in bacterial suppression. (4) Irradiation of GBS with or without NAD+ or NADH supplementation—two weak absorbers of 450 nm light—minimally suppressed GBS, indicating that a blue light-absorbing chromophore is essential for the bactericidal action of blue light. (5) Collectively, these findings show that in addition to the presence of a blue light-absorbing chromophore in bacteria, a chromophore with the right metabolic machinery and biochemical structure, capable of producing ROS, is necessary for 450 nm blue light to suppress GBS.

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

A study of concentration changes of Protoporphyrin IX and Coproporphyrin III in mixed samples mimicking conditions inside cancer cells for Photodynamic Therapy

Photodynamic Therapy (PDT) using Aminolevulinic acid (ALA) could be an effective and minimally invasively applicable way to treat many different types of tumors without radiation and large incisions by just applying a light pulse. However the PDT process is difficult to observe, control and optimize and the dynamical relationships between the variables involved in the process is complex and still hardly understood. One of the main variables affecting the outcome of the process is the determination of the interval of time between ALA inoculation and starting of light delivery. This interval, better known as drug-light interval, should ensure that enough Protoporphyrin IX (PPIX) is located in the vicinity of functional structures inside the cells for the greatest damage during the PDT procedure. One route to better estimate this time interval would be by predicting PPIX from the dynamical changes of its precursors. For that purpose, in this work a novel optical setup (OS) is proposed for differentiating fluorescence emitted by Coproporphyrin III (CPIII) and PPIX itself in samples composed of mixed solutions. The OS is tested using samples with different concentrations in mixed solutions of PPIX and the precursor CPIII as well as with a Polymethyl methacrylate test sample as additional reference. Results show that emitted fluorescence of the whole process can be measured independently for PPIX and its precursor, which can enable future developments on PPIX prediction from the dynamical changes of its precursor for subject-dependent drug-light interval assessment.

Fiber-optic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors – A preliminary study for improved Photodynamic Therapy applications

Abstract In this work we present first results of a laboratory manufactured filter-fluorometer to study differences in intensity and position of the main peaks of three porphyrins that appear during the Heme-Synthesis. Porphyrins play a major role in Photodynamic Therapy (PDT) for cancer treatment. Within the Heme-Synthesis, Porphyrins such as Protoporphyrin IX (PPIX) and its two precursors Coproporphyrin III (CPIII) and Uroporphyrin III (UPIII) represent photochemical agents that can interact with light to show fluorescence or generate Reactive Oxygen Species (ROS) to destroy cells. A major problem that arises is determining the ideal time slot to begin treatment after drug application. Our work is meant to show a way to solve this problem by looking at concentration changes of precursors appearing in Heme-Synthesis and using these changes to predict the occurence of PPIX inside the mitochondria.

A highly selective and sensitive LC-MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting peptide biomarkers.

Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug-drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials& methods:Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer.Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.14) were used as precursor ions to improve the assay sensitivity and selectivity over the singly charged precursor ions (m/z 655.28). Levels of CP-I and CP-III measured ranged 0.45-1.1 and 0.050-0.50ng/ml in plasma and 5-35 and 1-35ng/ml in urine, respectively. The described highly selective and sensitive CP-I/CP-III LC-HRMS assay offers options for earlier characterization and clinical safety projections for OATP1B1/3-mediated drug-drug interactions along with pharmacokinetic analyses of a new chemical entity as part of first-in-human clinical studies.

Measuring acne using Coproporphyrin III, Protoporphyrin IX, and lesion-specific inflammation: an exploratory study

Propionibacterium acnes: (P. acnes) produce Porphyrins; however, fluorescence measurement of Porphyrins from Ultraviolet-A (UVA) images has failed to establish a correlation. Acne clinical research and imaging has ignored the spectral excitation-emission characteristics and the exact pattern of the Porphyrins synthesized by P. acnes. In this exploratory study, for the first time, the possible relationships of Coproporphyrin III (CpIII) and Protoporphyrin IX (PpIX) fluorescence as well as acne lesion-specific inflammation measurements with clinical signs of acne are investigated. Furthermore, the sensitivity of these measurements in tracking and differentiating the known treatment effects of Benzoyl Peroxide (BPO) 5%, and combination of Clindamycin + BPO are also evaluated. Comedonal and papulopustular lesions identified by investigators during a live assessment of 24 mild-to-severe acne subjects were compared with fluorescence and inflammation measurements obtained from analysis of VISIA®-CR images. CpIII fluorescence spots showed a strong correlation (r = 0.69–0.83), while PpIX fluorescence spots showed a weak correlation (r = 0.19–0.27) with the investigators’ comedonal lesion counts. A strong correlation was also observed between the investigators’ papulopustular lesion counts and acne lesion-specific inflammation (r = 0.76). Our results suggest that CpIII fluorescence and acne lesion-specific-inflammation measurement can provide objective indication of comedonal and papulopustular acne severity, respectively. Furthermore, these measurements may be more sensitive and specific in evaluating treatment effects and early signs of acne lesion progression compared to investigators’ lesion counts.

Effects of plasma membrane ABCB6 on 5-aminolevulinic acid (ALA)-induced porphyrin accumulation in vitro: Tumor cell response to hypoxia

Currently, 5-aminolevulinic acid-based photodynamic diagnosis (ALA-PDD) is used to detect tumors during surgery and exploit tumor-specific accumulation of protoporphyrin IX (PpIX) after administration of ALA. In a recent study, we showed that the human ATP-binding cassette transporter ABCG2 plays a key role in the regulation of PpIX as a specific exporter. However, coproporphyrin III (CPIII) was also detected in urine after ALA administration in patients with tumor, indicating the presence of a CPIII transporter. We used two lines of human gastric cancer cells to measure the ALA-induced porphyrin metabolism. Intracellular and extracellular porphyrin levels and expressions of transporter were determined. In the present study, we showed that although ABCG2 did not transport CPIII, plasma membrane ABCB6 did. Moreover, under conditions of hypoxia, the expression of ABCB6 in plasma membrane was upregulated, resulting in increased extracellular CPIII concentrations. These data indicate that the expression of ABCB6 in plasma membrane is important for porphyrin accumulation after ALA administration, including hypoxic conditions.

Propionibacterium-produced coproporphyrin III induces Staphylococcus aureus aggregation and biofilm formation.

ABSTRACTThe majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera: Propionibacterium, Corynebacterium, and Staphylococcus. Among these staphylococci is the medically important bacterium Staphylococcus aureus. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium from Propionibacterium spp. induce S. aureus aggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associated Propionibacterium spp., as a cause of S. aureus aggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independent S. aureus biofilm development on an abiotic surface in multiple S. aureus strains. In strain UAMS-1, CIII stimulation of biofilm depended on sarA, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator of S. aureus aggregation and/or biofilm formation in the nostril or other sites inhabited by Propionibacterium spp. and S. aureus.

Porphyrins as urinary biomarkers for bladder cancer after 5-aminolevulinic acid (ALA) administration: The potential of photodynamic screening for tumors

Tumor biomarkers are commonly used for cancer screening and as indicators of treatment effects. We recently reported that urine porphyrin levels from tumor-bearing mice were elevated compared with those from normal mice after administration of 5-aminolevulinic acid (ALA). In the present study, we evaluated the use of urine samples from bladder cancer patients as tumor biomarkers. ALA, 1.0 g, was orally administered to 66 bladder cancer patients and 20 healthy adults. The urine concentrations of uroporphyrin I (UPI), uroporphyrin III (UPIII), coproporphyrin I (CPI), coproporphyrin III (CPIII), and total porphyrins were measured using High Performance Liquid Chromatography (HPLC) system. Almost all of the urinary porphyrin concentrations from the patients with bladder cancer were higher than those from healthy adults. Moreover, 8h after ALA administration, urinary UPI and CPI showed high sensitivity (100 for UPI and CPI) and specificity (96.4 for UPI and 91.4 for CPI). These results indicate that the presence of urinary porphyrins after administration of ALA may function as tumor biomarkers. This method represents a possible new tumor screening method called photodynamic screening (PDS) using ALA-induced porphyrins.

Comparative study of the bactericidal effects of 5-aminolevulinic acid with blue and red light on Propionibacterium acnes

Propionibacterium acnes naturally produces endogenous porphyrins that are composed of coproporphyrin III (CPIII) and protoporphyrin IX (PpIX). Red light alone and photodynamic therapy (PDT) improve acne vulgaris clinically, but there remains a paucity of quantitative data that directly examine the bactericidal effects that result from PDT on P. acnes itself in vitro. The purpose of this study was to measure the difference of bactericidal effects of 5-aminolevulinic acid (ALA)-PDT with red and blue light on P. acnes. P. acnes were cultured under anaerobic conditions and divided into two groups (ALA-treated group and control group), and were then illuminated with blue (415 nm) and red (635 nm) lights using a light-emitting diode (LED). The cultured P. acnes were killed with both blue and red LED light illumination. The efficacy increased with larger doses of light and a greater number of consecutive illuminations. We demonstrated that red light phototherapy was less effective for the eradication of P. acnes than blue light phototherapy without the addition of ALA. However, pretreatment with ALA could enhance markedly the efficacy of red light phototherapy.

Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins

We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM–PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN > PPIX > CPIII > CPI, which ranged from −22.2 to −6.1 kcal/mol. In general, the protein–tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components.

Urinary Porphyrin Excretion in Children is Associated with Exposure to Organochlorine Compounds

BackgroundHexachlorobenzene (HCB) and other organochlorines induce porphyria cutanea tarda (PCT) in animal studies. Evidence in humans, however, is contradictory. In neonates and adults from a population historically highly exposed to HCB (Flix, Catalonia, Spain), no relation with PCT or with porphyrin excretion was found.ObjectivesWe aimed to analyze the association between urinary porphyrin excretion and exposure to HCB and other organochlorinated compounds in children 4 years of age.MethodsOur birth cohort included all newborns from Flix and the five surrounding towns (where no airborne pollution occurred). Among the 68 children with porphyrins we measured in cord blood, 52 children 4 years of age provided blood to measure organochlorine compounds, hair for methylmercury, and urine for porphyrin excretion pattern.ResultsQuantitative porphyrin excretion was within the normal values. However, total porphyrins, coproporphyrin I (CPI), and coproporphyrin III (CPIII) adjusted to creatinine excretion increased with increasing levels of HCB, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p′-DDE), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p′-DDT), and polychlorinated biphenyl congener 153 (PCB-153). We found no association with methylmercury. When we fitted multiple pollutant models, p,p′-DDE had the strongest association. We found these associations in children from both Flix and other towns, and they were independent of breast-feeding and of organochlorine and porphyrin levels at birth.ConclusionHCB at current levels did not induce porphyria or increase uroporphyrins. However, the increase of urinary coproporphyrins suggests an incipient toxic effect of the organochlorines, especially for p,p′-DDE, on the hepatic heme-synthesis pathway that differs from the major effects seen in PCT.