The pigments in eggshell with different colour and the pigment regulatory gene expression in corresponding chicken's shell gland.

Eggshell colour is the unique appearance and economically valuable trait of eggs, whereas the colour is often short of uniformity, especially in the blue-shelled breeds, hence, their pigment differences and molecular mechanism need clarity. To investigate the relationship between the pigment content of eggshells and related gene expression in the eggshell glands of chickens, four subtypes of blue-shelled eggs ('Olive', 'Green', 'Blue', and 'Light') from the same blue-eggshell chicken line were selected; Hy-Line 'White' and 'Brown'-shelled eggs were used as control groups. The L*, a*, b* values, and protoporphyrin-IX and biliverdin contents in each group of eggshells were measured. In addition, the shell glands of the corresponding hens were collected to detect SLCO1B3 genotype and mRNA expression, and ABCG2 and HMOX1 transcription and protein expression. Eggshell colour L* values were negatively correlated with protoporphyrin-IX, b* values were positively correlated with total pigment content (P < 0.001), and a* values were positively correlated with protoporphyrin-IX (P < 0.001) but negatively with biliverdin. Moreover, all four blue-eggshell subtypes were SLCO1B3 homozygous, with SLCO1B3 mRNA expression in shell glands being significantly higher than in the White and Brown groups. ABCG2 and HMOX1 mRNA expression were highest in the Brown and Green groups, respectively (P < 0.05), and were positively correlated with protoporphyrin-IX (P < 0.001) and biliverdin contents in eggshells, respectively. Western blot and immunohistochemical results demonstrated that the Brown group had the highest ABCG2 expression (P < 0.05), followed by the Green and Olive groups. HMOX1 protein expression was higher in the Olive and Green groups (P < 0.05), and lowest in the White group. This study suggests that ABCG2 and HMOX1 have important regulatory roles in the production and transport of protoporphyrin-IX and biliverdin in blue-shelled chicken eggs, respectively.

Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Characterization of autofluorescence and quantitative protoporphyrin IX biomarkers for optical spectroscopy-guided glioma surgery.

5-Aminolevulinic acid (5-ALA)-mediated fluorescence does not effectively depict low grade gliomas (LGG) or the infiltrative tumor portion of high-grade gliomas (HGG). While spectroscopy improves sensitivity and precision, this is currently limited by autofluorescence and a second protoporphyrin IX (PpIX) fluorescence state at 620 nm. We investigated the autofluorescence to better characterize the present spectra and thus increase PpIX quantification precision and sensitivity. This study included 128 patients undergoing surgery for malignant glioma. 5-ALA (Gliolan) was administered before anesthesia, and fluorescence was measured using a hyperspectral device. It was found that all 2692 measured spectra consisted of contributions from 620 to 634 nm PpIX, NADH, lipofuscin, and flavins. The basis spectra were characterized and their use in spectral unmixing led to 82.4% lower fitting error for weakly fluorescing areas (p < 0.001), and 92.3% fewer false positive tumor identifications in control measurements (p = 0.0065) compared to previous works. They also decreased the PpIX620 contribution, thus halving the mean Ratio620/634 (p < 0.001). The ratio was approximately 0 for HGGs and increasing for LGGs, as demonstrated previously. Additionally, the Ratio620/634, the MIB-1/Ki-67 proliferation index, and the PpIX peak blue-shift were found to be significantly related to WHO grade, fluorescence visibility, and PpIX contribution (p < 0.001), and the value of these three as quantitative biomarkers is discussed.

Afterglow Amplification for Fast and Sensitive Detection of Porphyria in Whole Blood.

Porphyria is a group of genetic photodermatoses that cause too much porphyrin to accumulate in the blood, skin, and liver, resulting in skin photosensitivity and damage, liver disease, or potential liver failure. Conventional detection methods include high-performance liquid chromatography and fluorescence spectrometry. However, these methods usually require complicated pretreatment and time-consuming processes. Therefore, efficient and fast detection of porphyria is urgently needed. Herein, we develop a molecular afterglow reporter-based sensing scheme for the detection of porphyrins in whole blood. The afterglow reporter can respond to the production of singlet oxygen (1O2) of porphyrins after light excitation, and the detection signals can be amplified through adjusting the amount of singlet oxygen and afterglow reporter molecules. Moreover, without the use of a real-time excitation source, afterglow signals can avoid the scattering and autofluorescence interference in biological samples, thereby reducing background noise. More importantly, we prove the applicability of the afterglow reporter in the quantitative detection of porphyrins in whole blood and demonstrate its great clinical potential.

5-Aminolevulinic acid-mediated photodynamic activity in patient-derived cholangiocarcinoma organoids.

BACKGROUND: Accurate diagnosis of the disease extension of cholangiocarcinoma (CCA) is often difficult in clinical practice. The diagnostic yield of conventional pre-operative imaging or endoscopic procedures is sometimes insufficient for the evaluation of longitudinal spreading of CCA. Here we investigated the usefulness of 5-aminolevulinic acid (5-ALA) for the pre- or intra-operative diagnosis of CCA, using patient-derived organoids. METHODS: Four CCA- and two adjacent tissue-derived organoids were established. After 5-ALA treatment, we assessed their photodynamic activity using fluorescence microscopy. RESULTS: CCA organoids established from different patients showed diverse morphology in contrast to monolayer structures of non-tumor organoids, and had the ability to form subcutaneous tumors in immunodeficient mice. CCA organoids demonstrated remarkably high photodynamic activity based on higher accumulation of protoporphyrin IX as a metabolite of 5-ALA compared to non-tumor organoids (40-71% vs. < 4%, respectively). Importantly, cancer cell-specific high photodynamic activity distinguished the organoids originated from biliary stenotic lesions from those of non-stenotic lesions in a CCA patient. The high photodynamic activity did not depend on the expression profile of heme biosynthesis genes. CONCLUSIONS: Distinct 5-ALA-based photodynamic activity could have diagnostic potential for the discrimination of CCA from non-tumor tissues.

Organochlorine pesticides and polychlorinated biphenyls in common kestrel eggs from the Canary Islands: Spatiotemporal variations and effects on eggshell and reproduction.

Organochlorine (OC) pesticides were widely used on the Canary Islands (Spain) for intensive crop production and against plagues of African locust. A previous study performed in 1988-1994 showed a high concentration of p,p'-DDE in the eggs of common kestrels (Falco tinnunculus) from the island of Tenerife. The present study shows OC pesticide and polychlorinated biphenyl (PCBs) levels in 40 unhatched common kestrel eggs collected from southern Tenerife between 2009 and 2016. The protoporphyrin IX in eggshells has also been analysed in order to explore the use of this pigment as a biomarker. Egg biometry, status of embryo development, eggshell thickness and mass of extractable lipids of each egg were recorded. Surrounding land use and reproductive parameters (hatching and fledging rates) were obtained for each nest. The most abundant compound was p,p'-DDE (15.0 µg/g d.w), followed by PCBs (0.46 µg/g d.w.). The decline in p,p'-DDE levels in southern Tenerife (with 23.6 µg/g d.w. in 1988-1994) was 36.4%. p,p'-DDE levels were positively associated with the surface of active and abandoned cropland in a 200 m-radius around the nest and with proximity to urban areas. PCB levels were associated with proximity to roads. Shell thickness was negatively affected by the p,p'-DDE concentration. The concentration of protoporphyrin IX in the eggshell was negatively associated with the concentration of hexachlorobenzene in the egg content. Despite the total ban on the use of p,p'-DDT in Spain since 1986, p,p'-DDE levels remain elevated in those areas in which that use was formerly intensive.

Effects of post mortem pH and salting time on Zinc-protoporphyrin content in nitrite-free Serrano dry-cured hams.

There is a growing demand for clean label products and thus the elimination of curing additives in various dry-cured meats is of interest while maintaining colour characteristics. This study was aimed to examine the effect of pH at 24 h post mortem (pHSM24h ≤ 5.4; 5.4 > pHSM24h < 5.9; pHSM24h ≥ 5.9) and salting time (standard vs reduced) on zinc-protoporphyrin content, heme content and other physicochemical parameters of Serrano dry-cured hams manufactured without the addition of curing agents. Results showed that in those hams with higher post mortem pH heme content was increased whereas ZnPP content and proteolysis index were decreased. Reduced salting time decreased salt content whereas ZnPP and heme contents remained unaffected. Lower post mortem pH and reduced salting time led to a higher content in various free fatty acids which, in turn, were found to correlate positively with ZnPP formation. However, the observed changes in heme and ZnPP contents had no effect on the instrumental color of the final product.

Objective assessment of intraoperative tumor fluorescence reveals biological heterogeneity within glioblastomas: a biometric study.

PURPOSE: Heterogeneity within GBMs and variability of visualized fluorescence combine to confer practical limitations to the technique of optical imaging. A biometric analysis was planned to objectively ascertain and analyse this phenomenon METHODS: 25 adult glioblastoma subjects undergoing resection were prospectively accrued. Biopsies were taken from various parts of the tumor and safe peritumoral zones. White light (WL) and visualized fluorescence was subjectively recorded. Corresponding histopathology [coalescent (C) or infiltrating (I) tumor] and protoporphyrin-IX (PPIX) levels were assayed. RESULTS: WL was very sensitive for detecting tumor. SF was more specific and had high positive predictive value for detecting tumor. WF on the other hand had a poor discriminatory efficacy. Mean PPIX levels were 3.0, 2.01 and 0.16 for SF, WF, and NF respectively. WF had a wide variable range of PPIX levels. Within the coalescent tumor areas, there was a variable distribution of fluorescence (both subjective as well as objective PPIX levels) with only 54% samples showing SF and high PPIX. In seven cases this discordance was noted within the same tumor (biological heterogeneity). CONCLUSIONS: Fluorescence may miss important tumor areas even if objective assessment is used. Histologically similar tumor areas may exhibit contrasting fluorescence properties, a phenomenon which needs further investigation and elucidation of underlying mechanisms which could potentially be manipulated to optimize the utility of fluorescence guidance.

Characterization of Protoporphyrin IX Species in Vitro Using Fluorescence Spectroscopy and Polar Plot Analysis.

Protoporphyrin IX (PPIX) is a photodynamic therapy (PDT) agent for the treatment of various types of cancer. The effectiveness of PDT is believed to be associated with aggregation of PPIX in cells. However, the aggregation equilibrium of PPIX in the cellular environment and in solution is still poorly understood. This is attributed by the lack of a method that allows for controllable generation of PPIX aggregates and robust analysis technique for measuring their photophysical properties. In this study, the dynamics of PPIX aggregation were investigated under high pressure and different solvent conditions using time-resolved fluorescence spectroscopy. The data were analyzed on a polar plot, a model-free analysis method that has become increasingly popular for fluorescence lifetime studies. We discovered that increasing hydrostatic pressure enhanced the formation of J-type aggregates based on measured absorbance, spectra, and lifetime features. Formation of large aggregates, which have a subnanosecond lifetime in the excited state, was observed under the increasing concentration of divalent cations as well as under a solvent of around neutral pH. PPIX monomerizes from the aggregate as pH becomes more basic, not dimerization as proposed by previous studies. Here, we demonstrate that the combination of time-resolved measurement and polar plot analysis is very robust for monitoring the presence of different types of PPIX aggregates formed in various chemical environments.

High-Throughput Imaging of PPIX Using Confocal Microscopy.

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may cause toxicity or even provide utility in the diagnosis or treatment of cancers that have elevated cellular PPIX levels. This chapter describes a high-throughput plate-based imaging technique for determining cellular protoporphyrin levels by using the GE Healthcare InCell 6000 confocal imaging system to detect the presence and location of PPIX in each cell and may be adapted for use with other imaging systems. Laser excitation and a scientific-grade complementary metal oxide semiconductor (CMOS) camera generate short exposure times, decreasing photobleaching in the target cells that may result in inaccurate measurements of PPIX and increasing screening throughput. Nuclear staining was detected by using a laser with 405-nm excitation and 455-nm emission wavelengths, and the presence of PPIX was measured using 405-nm excitation and 706-nm emission wavelengths. Image analysis involving top-hat segmentation on both nuclear and PPIX staining was performed by using the InCell Analyzer Workstation software. This assay may be adapted to screen for PPIX formation, degradation, and transportation effectors. Indeed, the inclusion of PPIX transport inhibition would be expected to further widen the linear range of fluorescence and improve the method.

Uso de HFR-supra para el tratamiento de porfiria cutánea tarda en paciente en hemodiálisis crónica / Use HFR-supra for porphyria cutanea tarda treatment in hemodialysis patient

No disponible

Investigation of the post mortem zinc protoporphyrin IX fluorescence with respect to its protein-bound and unbound occurrence in aqueous meat extracts.

Zinc protoporphyrin IX (ZnPP) is known to accumulate in most meat products during storage. However, the pathway of its formation is not yet completely clarified. To gain more insights into the specificity of ZnPP occurrence, a SEC-HPLC-UV-fluorescence setup was established to screen the proteins in aqueous meat extracts for their ZnPP fluorescence during incubation. In accordance with previous studies it was identified by SDS-PAGE and MALDI-TOF-MS that ZnPP formation takes place in myoglobin. In this study, valuable new insights into the ZnPP forming pathway were gained, as our results indicated that a significant part of ZnPP - after being formed within the protein - is transitioned into free ZnPP during incubation. Additionally, the obtained results implied that ZnPP may also occur in proteins of higher molecular weight (>100 kDa).

Non-invasive measurement of erythrocyte zinc protoporphyrin in children.

BACKGROUND: Reliable screening for iron deficiency (ID) has required a blood sample and cost-intensive laboratory measurements. A novel method to non-invasively measure erythrocyte zinc protoporphyrin (ZnPP), an established marker for ID, is evaluated in children. METHODS: ZnPP was determined non-invasively by fluorescence measurements on the wet vermillion of the lower lip in 99 hospitalized children aged 9 months to 5 years. For comparison, conventional ID parameters and ZnPP were determined from blood samples. RESULTS: The non-invasively measured ZnPP values had limits of agreement (LoA) of 14 µmol ZnPP/mol heme (95% confidence interval: 9-20) compared to fluorescence measurements directly in blood. Repeated high-performance liquid chromatography reference determinations had comparable LoA of 14 µmol ZnPP/mol heme (9-17). Non-invasive ZnPP measurements had sensitivity and specificity of 67% (39-88%) and 97% (91-99%), and negative and positive predictive value of 94% (90-97%) and 80% (55-93%), for detecting ID as defined by the soluble transferrin receptor (sTfR). In groups with more severe ID as defined by serum ferritin and sTfR, higher ZnPP values were found, with the highest ZnPP values for the group with ID anemia. CONCLUSION: Non-invasive ZnPP measurements are reliably feasible in children. The simple and fast method has the potential to enable wide-spread screening for ID.

Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching.

Protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (5-ALA) is increasingly used as a fluorescent marker for fluorescence-guided resection of malignant gliomas. Understanding how the properties of the excitation light source and PpIX fluorescence interact with the surgical microscope is critical for effective use of the fluorescence-guided tumor resection technique. In this study, we performed a detailed assessment of the intensity of the emitted blue light and white light and the light beam profile of clinical grade operating microscopes used for PpIX visualization. These measurements revealed both recognized fluorescence photobleaching limitations and unrecognized limitations that may alter quantitative observations of PpIX fluorescence obtained with the operating microscope with potential impact on research and clinical uses. We also evaluated the optical properties of a photostable fluorescent standard with an excitation-emission profile similar to PpIX. In addition, we measured the time-dependent dynamics of 5-ALA-induced PpIX fluorescence in an animal glioma model. Finally, we developed a ratiometric method for quantification of the PpIX fluorescence that uses the photostable fluorescent standard to normalize PpIX fluorescence intensity. This method increases accuracy and allows reproducible and direct comparability of the measurements from multiple samples.

Photodynamic diagnosis with methyl-5-aminolevulinate in squamous intraepithelial lesions of the vulva: Experimental research.

The incidence of the High-grade Squamous Intraepithelial Lesion of the vulva, formerly vulvar intra-epithelial neoplasia is progressively increasing. Today, an early detection and a precise localization of vulvar lesions are still problematic issues, due to the lack of accuracy of the available diagnostic tool. A new approach is the photodynamic diagnosis based on the fluorescence detection of protoporphyrin IX (PpIX) in cancer cells after topical application of a cream of methyl amino-levulinic acid. This study aimed to evaluate the effectiveness of photodiagnosis in order to discriminate the intensity of PpIX fluorescence between vulvar tumor and healthy skin. After topical application of the cream, the fluorescence on xenografted A431 tumor and adjacent skin was non-invasively measured with optical fiber. The tumor to skin fluorescence ratios were 1.38 and 1.41 at respectively 3h and 6h after application, which were significantly higher compared to those observed before application. PpIX accumulation at different depths of the tumor was investigated by spectrofluorimetry after PpIX chemical extraction from tumor sections at 3h and 6h post-application. It was noticed at both application times that the concentration of PpIX within the tumor progressively decreased. However PpIX fluorescence was always detectable up to 2.5 mm, a depth equivalent to more than three quarters of the tumor. The tumor to exposed skin ratios of PpIX fluorescence showed a good selectivity up to1mm depth at 3h post-application and up to 1.5mm at 6h post-m-ALA. Thus, the photodynamic diagnosis using in vivo topical methyl amino-levulinic acid appears to be a promising way to detect the intraepithelial lesions of the vulva. Our results open the possibility for implementation of topical methyl amino-levulinic acid in clinical settings for recognition of vulvar high-grade squamous intraepithelial lesions.

Spectrophotometric photodynamic diagnosis of prostate cancer cells excreted in voided urine using 5-aminolevulinic acid.

To evaluate the feasibility of photodynamic diagnosis using 5-aminolevulinic acid (PDD-ALA) for detection of prostate cancer (PCa) cells in urine samples after prostate massage in patients who were suspected to have PCa. One hundred and eighty-nine patients with abnormal digital rectal examination and/or an elevated prostate-specific antigen (PSA) level who underwent initial prostate biopsy were recruited. After prostate massage, the first 60 mL of voided urine was collected. For PDD-ALA, 50 mL was used. The rest of collected urine was used for polymerase chain reaction (PCR) of PSA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). After incubation for 2 h, the intensity was measured at 635 nm under a 405-nm wavelength excitation. The results of PDD-ALA were compared with those of an initial transrectal ultrasound (TRUS)-guided prostate biopsy. Overall, 126/189 (67%) samples that showed bands of both PSA and GAPDH on PCR in urine samples were analyzed. The area under the curve, sensitivity, and specificity of PDD-ALA were 0.74, 77, and 67%, respectively. The value of PDD-ALA was significantly higher in patients with Gleason scores of 6 (p = 0.03), 7 (p = 0.005), and 8-10 (p = 0.0002) than in those with negative biopsy results. In the multivariate analysis, high PSA density, abnormal findings on TRUS, and a high value of PDD-ALA were significant markers for prediction of positive biopsy results. PDD-ALA was useful to predict positive biopsy results in patients who underwent initial prostate biopsy with suspected PCa. This PCa-detection method has potential for clinical use.

Assessment of the bacterial impact on the post-mortem formation of zinc protoporphyrin IX in pork meat.

The post-mortem accumulation of the heme biosynthesis metabolite zinc protoporphyrin IX (ZnPP) in porcine muscle is associated with both a meat-inherent and a bacterial enzymatic reaction during meat storage. To estimate the bacterial impact on ZnPP formation, meat and meat-like media were investigated by HPLC-FLD (and MALDI-TOF-MS) after inoculation with a representative microorganism (P. fluorescens). Results indicate the principal ability of meat-inherent bacteria to form ZnPP in meat extracts and meat-like media, but not on the meat muscle. Thus it was concluded that the ZnPP formation in meat is due to a meat-inherent enzymatic reaction induced by porcine ferrochelatase (FECH), while the bacterial (FECH) induced reaction seems to be not significant.

Scanning Fiber Endoscope Improves Detection of 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence at the Boundary of Infiltrative Glioma.

OBJECTIVE: Fluorescence-guided surgery with protoporphyrin IX (PpIX) as a photodiagnostic marker is gaining acceptance for resection of malignant gliomas. Current wide-field imaging technologies do not have sufficient sensitivity to detect low PpIX concentrations. We evaluated a scanning fiber endoscope (SFE) for detection of PpIX fluorescence in gliomas and compared it to an operating microscope (OPMI) equipped with a fluorescence module and to a benchtop confocal laser scanning microscope (CLSM). METHODS: 5-Aminolevulinic acid-induced PpIX fluorescence was assessed in GL261-Luc2 cells in vitro and in vivo after implantation in mouse brains, at an invading glioma growth stage, simulating residual tumor. Intraoperative fluorescence of high and low PpIX concentrations in normal brain and tumor regions with SFE, OPMI, CLSM, and histopathology were compared. RESULTS: SFE imaging of PpIX correlated to CLSM at the cellular level. PpIX accumulated in normal brain cells but significantly less than in glioma cells. SFE was more sensitive to accumulated PpIX in fluorescent brain areas than OPMI (P < 0.01) and dramatically increased imaging time (>6×) before tumor-to-background contrast was diminished because of photobleaching. CONCLUSIONS: SFE provides new endoscopic capabilities to view PpIX-fluorescing tumor regions at cellular resolution. SFE may allow accurate imaging of 5-aminolevulinic acid labeling of gliomas and other tumor types when current detection techniques have failed to provide reliable visualization. SFE was significantly more sensitive than OPMI to low PpIX concentrations, which is relevant to identifying the leading edge or metastasizing cells of malignant glioma or to treating low-grade gliomas. This new application has the potential to benefit surgical outcomes.