Heme Precursor Research Articles

Biomarkers of exposure and effect in a working population exposed to lead, manganese and arsenic

ABSTRACT Lead (Pb), manganese (Mn) and arsenic (As) are among the major toxicants in mining environments. Miners are commonly and repeatedly exposed to this toxic mixture. Some adverse effects may appear at concentrations below environmental quality guidelines for individual mixture components. Further, Pb, Mn, and As induce common adverse outcomes, such as interferences in the cholinergic system and heme synthesis. It is thus vital to monitor miners through biomarkers (BM), such that subclinical effects may be identified at an early stage. The main objectives of this study were to evaluate the exposure of a mining population to these three metals and determine alterations in cholinergic and heme synthesis parameters. Blood and urine samples of workers (n = 60) were obtained from a Portuguese mining industry and compared with a control population (n = 80). The levels of the metals were determined in biological samples, as well as urinary heme precursor levels, delta aminolevulinic acid (ALA) and porphyrins, and blood acetylcholinesterase (AChE) activity. The miners exhibited significantly higher values of Pb and As in blood and urine compared to control. In the case of Mn near or slightly higher than limit values were found. Our data show that heme precursors may be used simultaneously with metal levels as BMs for multiple metal exposures on an individual basis, resulting in 94.3% and 95.7% accuracy, respectively, in blood and urine, for subjects correctly identified with respect to occupation. This study also revealed that biological monitoring of this working population regarding metal body burden and heme precursor accumulation is advisable.

Effect of 5-aminolevulinic acid on the expression of carcinogenesis-related proteins in cultured primary hepatocytes.

Acute intermittent porphyria (AIP) is a heme pathway disorder caused by a decrease in the activity and synthesis of porphobilinogen deaminase. Thus, the first heme precursor 5-aminolevulinic acid (ALA) accumulates in the liver. Reactive oxygen species (ROS) resulting from ALA oxidation may be correlated to a higher incidence of hepatocellular carcinoma (HCC) in AIP patients. However, the molecular mechanisms of this relationship have not been thoroughly elucidated to date. In this study, we investigated the effect of increasing levels of ALA on the expression of proteins related to DNA repair, oxidative stress, apoptosis, proliferation and lipid metabolism. Primary rat hepatocytes were isolated by the collagenase perfusion method, lipoperoxidation was evaluated by a TBA fluorimetric assay and Western blotting was used to assess protein abundance. The data showed that ALA treatment promoted a dose-dependent increase of p53 expression, downregulation of Bcl-2, HMG-CoA reductase and OGG1 and an increase in lipoperoxidation. There was no alteration in the expression of the transcription factor NF-κB, catalase and superoxide dismutase. ALA oxidation products induced protein regulation patterns, suggesting the interconnection of cellular processes, such as the intrinsic pathway of apoptosis, redox homeostasis, cell proliferation, lipid metabolism and DNA repair. This study helps to elucidate the molecular mechanisms of hepatotoxicity mediated by ALA pro-oxidant effects and supports the hypothesis that ALA accumulation correlates with a higher incidence of hepatic carcinogenic events.

The association between chemical-induced porphyria and hepatic cancer.

The haem biosynthetic pathway is of fundamental importance for cellular metabolism both for the erythroid and nonerythroid tissues. There are several genetic variants of the pathway in the human population that cause dysfunction of one or other of the enzymes resulting in porphyrias of varying severity. Serious chronic hepatic and systemic diseases may result. Some of these can be precipitated by exposure to drugs including hormones, barbiturates and antibiotics, as well as alcohol and particular chlorinated aromatic chemicals. In experimental animals some of the steps of this pathway can also be severely disrupted by a variety of environmental chemicals, potential drugs and pesticides, especially in the liver, leading to the accumulation of uroporphyrins derived from the intermediate uroporphyrinogens or protoporphyrin IX, the immediate precursor of haem. With some of these chemicals this also leads to cholestasis and liver cell injury and eventually hepatic tumours. The review evaluates the available evidence linking hepatic porphyria with carcinogenesis in naturally occurring human genetic conditions and in chemically-induced porphyrias in laboratory animals. The existing data showing gender, strain, and species differences in sensitivity to the chemical-induced porphyrias, liver injury and liver tumours are discussed and the role that transgenically altered mouse models have played in defining the varying mechanisms. Finally, the review proposes a novel, unifying hypothesis linking the hepatotoxicity induced by the accumulation of various porphyrins, with the increased risk of developing hepatic cancer as a long term consequence.

Characterization and origin of heme precursors in amniotic fluid: lessons from normal and pathological pregnancies.

Heme is the prosthetic group of numerous proteins involved in vital processes such as oxygen transport, oxidative stress, and energetic mitochondrial metabolism. Free heme also plays a significant role at early stages of development and in cell differentiation processes. The metabolism of heme by the fetal placenta unit is not well-established in humans. In a retrospective study, we measured heme precursors in the amniotic fluid (AF) of 51 healthy women, and 10 AF samples from pregnancies with either upper or lower intestinal atresia or ileus were also analyzed. We showed that the porphyrin precursors aminolevulinic acid, porphobilinogen, and protoporphyrin IX are present at the limit of detection in the AF. Total porphyrin levels decreased progressively from week 13 to week 33 (p < 0.01). Interestingly, uroporphyrin, initially detected as traces, increased with maturation, in contrast to coproporphyrin. Uro- and coproporphyrins were type I immature isomers (>90%), suggesting a lack of maturity in the fetal compartment of the heme pathway. Finally, the differential analysis of AF from normal and pathological pregnancies demonstrated the predominant hepatic origin of fetal porphyrins excreted in the AF. This study gives the first insight into heme metabolism in the AF during normal and pathological pregnancies.

P-99 - 5-Aminolevulinic acid induced apoptosis via oxidative stress in normal gastric epithelial cells

5-Aminolevulinic acid (5-ALA), a precursor of heme, is utilized in a variety of applications including cancer treatment, surgery, and plant nutrition. However, 5-ALA itself induces oxidative stress and subsequent lipid peroxidation. Reactive oxygen species (ROS) are factors in oxidative stress, not only causing cellular injury but also inducing several signal transduction pathways. Especially in cancer cells, a significant amount of signaling activation and subsequent activation of protein is caused by the enhancement of ROS production. ROS levels in normal cells are low and an oxidative condition is harmful; hence, administration of 5-ALA to normal cells may induce oxidative stress, resulting in cell death. In this study, we investigated the effect of 5-ALA on normal and cancer cells with regard to oxidative stress. We used the rat normal gastric cell line RGM and its cancer-like mutant cell line RGK. 5-ALA treatment of RGM cells enhanced ROS generation and induced apoptosis associated with p53, whereas RGK cells were unaffected. In addition, RGM cell viability was recovered by application of N-acetyl-L-cysteine or p53 inhibitor. These results suggest that 5-ALA causes oxidative stress in normal gastric cells and induces apoptosis via the p53-dependent pathway.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells.Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted.Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model.Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Abstract 21202: 5-Aminolevulinic Acid Combined With Sodium Ferrous Citrate Ameliorates Post Cardiac Ischemia/Reperfusion Injury via Regulating Inflammatory Cytokines

Post ischemia/reperfusion injury (I/R) reduces beneficial effect of restoration of coronary flow in the setting of acute myocardial infarction. Heme oxygenase-1 (HO-1) is inducible enzyme, and plays a crucial role in reaction against for oxidative stress, inflammatory, and apoptosis. Previous studies reported that HO-1 could reduce the infarct size after ischemia/reperfusion injury (I/R). 5-aminolevulinic acid (5-ALA) is known to be a naturally occurring metabolic precursor of heme, and 5-ALA combined with ferrous iron can induce HO-1 in various cells. In this study, we investigate the protective effect of 5-ALA pretreatment after I/R using mice model. Male C57/BL 6J mice (8-12 weeks of age and weighing 21-26 g) were pretreated with 100 mg/kg 5-ALA/HCL and 157 mg/kg sodium ferrous citrate or placebo 48, 24, 1 h before I/R, and underwent 50 minutes of left coronary artery occlusion followed by 24 h reperfusion. Infarct area (IA), area at risk (AAR), and area of left ventricle (LV) were determined by 5% Evans blue and 1% triphenyltetrazolium chloride (TTC) double staining after 24h reperfusion (Figure [A]). Pretreatment of 5-ALA group significantly reduced IA/AAR compared to the placebo group (34.0% [28.1 - 37.6%] VS 51.7% [45.8 - 54.0%], p= 0.001, n=6, Figure [B and C]). Real time PCR assay after 24h reperfusion showed that expression of mRNA of TNF-α (p=0.040), IL-1β(p=0.024), MMP-9 (p=0.011) and BNP (p=0.046) were significantly lower in 5-ALA group than that in placebo group. These findings suggested that 5-ALA pretreatment regulated inflammatory reaction after I/R. The beneficial effect of 5-ALA could be related with HO-1 induction. In conclusion, pretreatment of 5-ALA combined with ferrous iron could play a protective role after I/R by attenuating inflammatory reaction.

Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli

One of the main threats to the achievements in modern medicine is antimicrobial resistance. Molecular targeting of bacterial acquisition mechanisms of heme has been suggested to be an alternative to antibiotics. In the present study, HPLC-MS/MS combined with a simple clean-up based on liquid-liquid extraction has been developed and evaluated for simultaneous determination of heme and porphyrin heme precursors in microorganisms. Experimental design was used to optimize the extraction parameters, to obtain a method with high recovery, low matrix effects, and high precision. The effects of additives in the culture medium on the biosynthesis of heme were studied using Escherichia coli as a model microorganism. 5-Aminolaevulinic acid and hemin increased the heme concentration in E. coli by a factor of 1.5 and 4.5, respectively. Addition of 5-aminolaevulinic acid bypassed the E. coli negative feedback control of heme biosynthesis, which led to high amounts of intracellular porphyrins. The high heme concentration obtained when hemin was used as a culture additive shows that E. coli has an uptake of heme from its surroundings. In contrast, addition of cobalt protoporphyrin IX to the growth medium reduced the amount of heme in E. coli, demonstrating this compound’s ability to mimic real heme and inhibit the heme acquisition mechanisms.

Abstract 5842: Therapeutic enhancement of aminolevulinic acid-mediate tumor fluorescence and photodynamic therapy with kinase inhibitors

Abstract Photodynamic therapy (PDT) involves the combination of a photosensitizer and light of a specific wavelength. Upon light activation in the presence of oxygen, photosensitizer molecules generate reactive oxygen species that cause cytotoxicity by inducing oxidative stress. Aminolevulinic acid (ALA) is a pro-drug used for the diagnosis and PDT treatment of various solid tumors based on endogenous production of heme precursor protoporphyrin IX (PpIX). Although all human cells express heme biosynthesis enzymes and produce PpIX, tumor cells are often found to have higher PpIX production and accumulation than normal cells, allowing for the detection and treatment of solid tumors. In exploring ALA-based tumor detection and therapy in breast cancer, we have reported that high ABCG2 transporter activity in triple negative breast cancer cells (TNBC) contributes to reduced PpIX levels in tumor cells, causing cell resistance to ALA-PDT. The administration of an ABCG2 inhibitor Ko143 was able to reverse cell resistance to ALA-PDT by increasing PpIX levels. In this study, we showed that some kinase inhibitors were able to increase ALA-mediated PpIX by inhibiting ABCG2 activity. Particularly, our results demonstrate that lapatinib, a dual inhibitor of EGFR and Her2, increased the ALA-PpIX level and cell sensitivity to ALA-PDT in the MDA-MB-231 TNBC cells. Lapatinib treatment had little effect on MCF10A breast epithelial cells. These results indicate that small molecule kinase inhibitors such as lapatinib can be used for enhancing ALA-based tumor detection and PDT in tumors with high ABCG2 activity. Citation Format: Bin Chen, Pratheeba Palasuberniam. Therapeutic enhancement of aminolevulinic acid-mediate tumor fluorescence and photodynamic therapy with kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5842. doi:10.1158/1538-7445.AM2017-5842

How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells

Numerous near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial photoreceptors but lack of their systematic comparison makes researcher's choice rather difficult. Here we evaluated side-by-side several modern NIR FPs, such as blue-shifted smURFP and miRFP670, and red-shifted mIFP and miRFP703. We found that among all NIR FPs, miRFP670 had the highest fluorescence intensity in various mammalian cells. For instance, in common HeLa cells miRFP703, mIFP, and smURFP were 2-, 9-, and 53-fold dimmer than miRFP670. Either co-expression of heme oxygenase or incubation of cells with heme precursor weakly affected NIR fluorescence, however, in the latter case elevated cellular autofluorescence. Exogenously added chromophore substantially increased smURFP brightness but only slightly enhanced brightness of other NIR FPs. mIFP showed intermediate, while monomeric miRFP670 and miRFP703 exhibited high binding efficiency of endogenous biliverdin chromophore. This feature makes them easy to use as GFP-like proteins for spectral multiplexing with FPs of visible range.

Accumulation of heme biosynthetic intermediates contributes to the antibacterial action of the metalloid tellurite

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.

Modulating heme biosynthesis, through the administration of exogenous aminolevulinic acid may attenuate monocrotaline induced‐pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease that initiates in small pulmonary arteries and arterioles, which undergo persistent vasoconstriction and vascular remodeling. This process elevates vascular resistance, pulmonary arterial pressure and ultimately results in right heart hypertrophy and failure. PAH can be induced in rats through exposure to the pneumotoxin monocrotaline (MCT). Deregulation of heme biosynthesis, which might accompany/contribute to elevated mitochondrial/cytosolic reactive oxygen species (ROS) is thought to propagate MCT‐induced PAH disease process. Elevated oxidative stress can augment the redox‐sensitive proto‐oncogene c‐Src kinase in PASMCs and thus might drive vascular remodeling further through the (c‐Src‐STAT3) axis. By administering the biosynthetic precursor of heme, aminolevulinic acid (ALA), we aim to drive heme biosynthesis and probably attenuate oxidative stress‐induced c‐Src activation.

Gallium-Protoporphyrin IX Inhibits Pseudomonas aeruginosa Growth by Targeting Cytochromes.

Pseudomonas aeruginosa is a challenging pathogen due to both innate and acquired resistance to antibiotics. It is capable of causing a variety of infections, including chronic lung infection in cystic fibrosis (CF) patients. Given the importance of iron in bacterial physiology and pathogenicity, iron-uptake and metabolism have become attractive targets for the development of new antibacterial compounds. P. aeruginosa can acquire iron from a variety of sources to fulfill its nutritional requirements both in the environment and in the infected host. The adaptation of P. aeruginosa to heme iron acquisition in the CF lung makes heme utilization pathways a promising target for the development of new anti-Pseudomonas drugs. Gallium [Ga(III)] is an iron mimetic metal which inhibits P. aeruginosa growth by interfering with iron-dependent metabolism. The Ga(III) complex of the heme precursor protoporphyrin IX (GaPPIX) showed enhanced antibacterial activity against several bacterial species, although no inhibitory effect has been reported on P. aeruginosa. Here, we demonstrate that GaPPIX is indeed capable of inhibiting the growth of clinical P. aeruginosa strains under iron-deplete conditions, as those encountered by bacteria during infection, and that GaPPIX inhibition is reversed by iron. Using P. aeruginosa PAO1 as model organism, we show that GaPPIX enters cells through both the heme-uptake systems has and phu, primarily via the PhuR receptor which plays a crucial role in P. aeruginosa adaptation to the CF lung. We also demonstrate that intracellular GaPPIX inhibits the aerobic growth of P. aeruginosa by targeting cytochromes, thus interfering with cellular respiration.

Pengaruh suplementasi seng dan vitamin B6 terhadap kadar hemoglobin, hematokrit dan indeks eritrosit pasien malaria vivax yang anemia

Background: Zinc supplementation decrease the risk of malaria parasitaemia, increasing ferritin serum level and hemoglobin in patients with malaria. Zinc influence the activity of aminolevulinic acid dehydratase (ALAD) an enzymes that catalizes heme synthesize. Vitamin B6 in pyridoxal 5-phosphate (PLP) from has a role in alpha-aminolevulinic acid (ALA) formation, which is the precursor of heme in hemoglobin. PLP also inhibit the growth of the malaria parasite.Objective: The objective was to analyze the effect of zinc and vitamin B6 supplementation on hemoglobin level, hematocrit and erythrocyte indexs (MCV, MCH and MCHC) of anemic Plasmodium vivax malaria patients.Methods: Double Blind Randomised Controlled Trial with pre and post test design. 30 subjects were divided into two groups: the suplementation group were given zinc 1x10 mg/day with vitamin B6 1x5 mg/day and the control group were given a placebo for 30 days. Data analysis by paired t-test, independent t-test and Mann Whitney.Results: There was an increase in Hb levels in the suplementation group (p=0.0001), the control group (p=0.001) and there was a significant difference on the increase between the two groups (p=0.020). Hematocrit significant increase only in the suplementation group (p=0.0001). There were no differences on erythrocyte index parameter in both groups.Conclusion: Zinc and vitamin B6 supplementation of for 30 days increase the hemoglobin level, hematocrit and there were no effect on erythrocyte indexs of anemic Plasmodium vivax malaria patients.

Biochemical Surrogate Markers of Hemolysis Do Not Correlate with Directly Measured Erythrocyte Survival in Sickle Cell Anemia

Introduction: Hemolysis has been proposed as a cause of several complications of sickle cell anemia (HbSS), including pulmonary hypertension, priapism, leg ulcers, and stroke. No human study of the role of hemolysis in these complications has directly quantified hemolysis; all used surrogate markers. We applied a recently validated stable isotope glycine red blood cell (RBC) labeling method (Am J Hematol. 2015;90:50-5) to measure RBC survival, a direct measure of the rate of hemolysis, to determine whether commonly used surrogate markers of hemolysis accurately estimate RBC survival in HbSS.Methods: Orally administered stable isotope-labeled glycine (15N-glycine), a metabolic precursor of heme, was used to track an age cohort of RBCs in participants with HbSS. The atomic excess of 15N in heme extracted from blood over time was monitored by mass spectrometry to calculate mean RBC survival. We also measured complete blood counts, reticulocyte counts, fetal hemoglobin (HbF), blood levels of biochemical surrogates of hemolysis (LDH, AST, bilirubin, and plasma free Hb), alpha-globin genotype, and tricuspid jet regurgitant velocity (TRV) by echocardiography.Results: There were 5 males and 6 females; mean age was 23 years (range 16-41). Two participants completed the study twice for a total of 13 labeling studies. Mean RBC survival was 31.9 days (S.D. 12.0; range 14.1-53.6). Mean RBC survival was inversely correlated with absolute reticulocyte count (ARC; r = -0.84, P < 0.001; Panel A) and percentage of reticulocytes (r = -0.78, P = 0.002). There was also a positive correlation between mean RBC survival and percent HbF (r = 0.58, P = 0.038; Panel B). The commonly used biochemical surrogate markers of hemolysis, AST, LDH, total bilirubin, indirect bilirubin, and plasma free Hb, were not correlated with directly measured RBC survival (Panels C & D). The "hemolytic index" or "hemolytic component" [derived from the combination of LDH, AST, total bilirubin and reticulocyte count (Blood. 2009;114:4639-44)] was not more strongly correlated with mean RBC survival than ARC (r = -0.73, P = 0.004). That is, the hemolytic component provided no more information about mean RBC survival than ARC alone. We found a correlation between TRV and LDH (r = 0.62, P = 0.03; Panel E), as previously described, but no correlation between TRV and directly measured RBC survival in the same participants (r = 0.09, P = 0.79; Panel F). This indicates that the relationship between TRV and LDH, while valid and reproducible, is not readily explained by hemolytic rate.Conclusions: RBC labeling with orally administered 15N-glycine is a safe and practical method to measure RBC survival directly, and thereby quantify hemolysis. Commonly used biochemical surrogate markers of hemolytic rate (LDH, AST, bilirubin, and plasma free Hb) did not correlate with directly measured RBC survival. These markers should be interpreted cautiously in HbSS. Only reticulocyte count and HbF level correlated with directly measured RBC survival. Although ARC appears to be a reasonable surrogate marker of hemolysis, it is indirect and explains only 70% of the variation in RBC survival. If greater accuracy is required for physiological studies or clinical trials, 15N-glycine RBC labeling can directly and accurately quantify hemolysis. [Display omitted] DisclosuresQuinn:Amgen: Research Funding; Eli Lilly: Research Funding; Silver Lake Research Corporation: Consultancy. Joiner:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Biochemical surrogate markers of hemolysis do not correlate with directly measured erythrocyte survival in sickle cell anemia.

Hemolysis is a key feature of sickle cell anemia (HbSS). Direct quantitation of hemolysis could be used as an objective outcome in clinical trials of new therapeutics for HbSS and would also enable better human studies of the pathogenesis of complications of HbSS that are ostensibly hemolysis-related, such as pulmonary hypertension. However, contemporary human studies in HbSS have used only surrogate markers of hemolysis rather than direct measurements of RBC survival. We directly quantified hemolysis in HbSS by measuring survival of an age cohort of RBCs labeled with a stable isotope, administered orally as 15 N-glycine, a metabolic precursor of heme. The atomic excess of 15 N in heme extracted from blood was monitored by mass spectrometry over time. We performed 13 labeling experiments in 11 individuals with HbSS. Mean RBC survival was 31.9 days (range 14.1-53.6). Both HbF level, a known determinant of hemolysis, and absolute reticulocyte count (ARC), an index of the marrow's response to hemolysis, correlated with directly measured RBC survival (r = 0.61, P < 0.002; r = -0.84, P < 0.001). However, commonly used biochemical surrogates of hemolysis (LDH, AST, bilirubin, and plasma free hemoglobin) did not correlate with directly measured RBC survival. These biochemical surrogates should be interpreted cautiously, at best, in clinical trials and human physiologic studies in HbSS. ARC was the best correlate of total hemolysis, but only 70% of the variation in RBC survival was reflected in this marker. If greater accuracy is required in human studies, 15 N-glycine RBC labeling can directly and accurately quantify hemolysis. Am. J. Hematol. 91:1195-1201, 2016. © 2016 Wiley Periodicals, Inc.

146 - The Exposure of 5-Aminolevulinic Acid Induced Apoptotic Cell Death via Oxidative Stress in Normal Gastric Epithelial Cells

Heme is an essential substance for oxygen metabolism. 5-aminolevulinic acid (ALA) is a precursor of heme and accelerates plants, hair and sperm growth. However, ALA is reported to be an oxidative stressor. We previously reported that reactive oxygen species (ROS) enhanced cancer cellular bioactivity such as metastasis and invasion. On the other hand, over-generation of ROS induces apoptotic cell death in normal cells. Thus, ROS caused by ALA may promote cancerous malignancy and give damage to normal cells. In this study, we investigated the different effect of ALA between normal cells and cancer cells using a rat normal gastric epithelial cell line, RGM and its chemically mutated cell line, RGK. 50 μM ALA for 48h induced normal cellular apoptosis, not in cancer cells. In addition, the normal cell death was suppressed by treatment with N-acetylcysteine. Therefore, we concluded that ALA induced ROS and subsequent normal cell specific death via oxidative stress. From this result, administration of ALA for cancer patients may bring getting worse.

Intermittently We Get Things Right: A Case of Porphyria After Esophagogastrectomy

Acute porphyrias are rare inherited metabolic disorders of heme production, intermittently presenting with abdominal pain and neurovisceral symptoms. We discuss the case of a 58-year-old African American female with a history of gastroparesis and esophageal adenocarcinoma, who presented with intractable abdominal pain, nausea and vomiting. Her symptoms were initially attributed to her previously established gastrointestinal diagnoses; however, an in-depth workup for gastric, hepatic, biliary and vascular causes was negative. Ultimately, a urine porphyria panel revealed elevated porphyrins and the patient was treated with a high carbohydrate diet and hemin infusion, with subsequent normalization of her urine porphyrins and improvement of her symptoms. Acute Porphyrias are exceedingly rare. However, the occurrence of acute porphyria after gastric surgery has been previously reported and attributed to diminished carbohydrate intake. Similarly, this patient's esophagogastrectomy reduced her caloric carbohydrate intake, perhaps resulting in the accumulation of heme precursors and the onset of a clinical crisis. The patient's dramatic normalization of urine porphyrins following hemin treatment underscores the validity of her diagnosis. Thus, we propose that acute porphyria should be considered in patients with recurrent abdominal pain, nausea and vomiting and a negative work-up for another intra-abdominal pathology. Porphyrias should also be considered in patients with persistent pain after abdominal surgery and those with reduced caloric intake. Early diagnosis of porphyria is key to effective treatment with hemin. Simple spot urine tests for urine porphinobilinogen, although not performed in this case due to low suspicion, can easily rule out the diagnosis, and should be the first step in the workup of such patients.Table 1: Urine porphyria panel results prior to hemin treatment and after hemin treatment. All values were initially elevated and were within normal levels after treatment. Urine ALA remained elevated 10 days after admission.

High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX

The heterologous overexpression level of the bacterial dye decolorizing peroxidase TfuDyP in Escherichia coli was increased sixty fold to approximately 200mg of purified enzyme per liter culture broth by fusing the enzyme to the small ubiquitin-related modifier protein (SUMO). The highly overexpressed SUMO-TfuDyP was, however, almost inactive. Analysis of the enzyme by UV–vis absorption spectroscopy and high-resolution mass spectrometry showed that a large fraction of the highly overexpressed enzyme contained the iron deficient heme precursor protoporphyrin IX (PPIX) instead of heme. Here we show that the activity of the enzyme was dependent on the expression level of the protein.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.