Total Heme Research Articles

Effect of Acute Hyperglycemia on Microvascular Hemodynamics and Tissue Oxygenation during Handgrip Exercise

Acute hyperglycemia elicits endothelial dysfunction at rest through reactive oxygen species-mediated damage to the endothelial surface layer (ESL). The ESL is associated with many of the mechanisms responsible for appropriate microvascular adjustments to exercise. PURPOSE: We tested the hypotheses that acute hyperglycemia would lead to 1) an ‘overshoot’ in deoxygenated heme concentration (deoxy-[heme]) at exercise onset reflecting greater fractional oxygen extraction and 2) less increase in total heme concentration (total-[heme]) during exercise reflecting less increase in microvascular hematocrit. METHODS: Three healthy young men (26 ± 4 yr) completed a 10-minute constant-load handgrip exercise test at 40% of peak power (9.6 ± 0.7 W) under control conditions (CON) and during acute hyperglycemia (HGL), i.e., 90-minutes after oral consumption of a 10 ounce solution containing 75g of dextrose. Near-infrared spectroscopy was used to measure deoxy-[heme] and total-[heme] of the flexor digitorum superficialis (FDS) continuously at rest and during exercise. RESULTS: Deoxy-[heme]CON and total-[heme]CON were significantly greater during exercise (189 ± 28 μM and 341 ± 34 μM, respectively) compared to rest (164 ± 13 μM and 302 ± 17 μM, respectively) (p < 0.01). Deoxy-[heme]HGL and total-[heme]HGL were significantly greater during exercise (181 ± 14 μM and 322 ± 27 μM, respectively) compared to rest (168 ± 26 μM and 313 ± 16 μM, respectively) (p < 0.01). As a percent of steady-state (SS) values, deoxy-[heme]CON was greater than deoxy-[heme]HGL from 35s until 75s of exercise (p<0.05). Deoxy-[heme]CON and deoxy-[heme]HGL were not different at SS. Total-[heme]HGL was significantly lower than total-[heme]CON at SS (p < 0.01). CONCLUSION: These data suggest that acute hyperglycemia leads to 1) increased fractional oxygen extraction at the onset of moderate-intensity handgrip exercise reflecting impaired perfusive oxygen delivery and 2) less of an increase in microvascular hematocrit reflecting impaired diffusive oxygen conductance during exercise steady state.

Oxygen Utilization During The Contraction-relaxation Of Isometric Knee Extension Exercise

PURPOSE: Sufficiently high intramuscular pressures during contraction can lead to increased resistance to perfusive oxygen delivery and therefore increased fractional oxygen extraction in order to maintain muscle VO2. Near-infrared spectroscopy (NIRS) has been used to estimate fractional oxygen extraction via deoxygenated heme concentrations (deoxy-[heme]) and changes in microvascular hematocrit via changes in total heme concentrations (total-[heme]). We tested the hypotheses that during severe and extreme intensities 1) deoxy-[heme] would decrease during contraction compared to relaxation and 2) total-[heme] would be unchanged during the contraction-relaxation cycle. METHODS: Four subjects (2 men, 24.0 ± 3.7 yrs, 81.9 ± 27.4 kg, 168.9 ± 10.2 cm) completed two isometric knee extension tests to failure at 40% (severe) and 70% (extreme) MVC. NIRS was placed on the right vastus lateralis to continuously measure deoxy-[heme] and total-[heme] during relaxation and contraction. Deoxy-[heme] and total-[heme] were averaged during the final 5 contraction-relaxation cycles and compared using paired t-tests. RESULTS: Average deoxy-[heme] during contraction and relaxation at 40% was 44.8 ± 31.8 υM and 44.4 ± 31.6 υM, respectively. Average deoxy-[heme] during contraction and relaxation at 70% was 41.9 ± 24.5 υM and 40.1 ± 23.6 υM, respectively. Average total-[heme] during contraction and relaxation at 40% was 127.0 ± 106.4 υM and 124.2 ± 105.2 υM, respectively. Average total-[heme] during contraction and relaxation at 70% was 116.0 ± 89.4 υM and 115.0 ± 90.5 υM, respectively. Although statistical significance was not detected (p=0.06), all four subjects demonstrated an increase in deoxy-[heme] at 70% MVC, but not at 40%. CONCLUSIONS: These data suggest that the contraction-relaxation cycle may result in fluctuations in fractional oxygen extraction during extreme but not severe isometric exercise. Furthermore, the contraction-relaxation cycle does not appear to alter microvascular hematocrit.

TECHNOLOGICAL AND NUTRITIVE CHARACTERIZATION OF “QUEIJO DE PORCO” SAUSAGE TRADITIONALLY PRODUCED AND COMMERCIALIZED IN SOUTHERN BRAZIL

The use of byproducts and retails from boning in meat products development is of great importance to the meat industry, as it adds value to a plentiful raw material and provide an increase in the protein content of formulated products. In Brazil, the manufacturing of traditional products based on byproducts like “Queijo de Porco” is not standardized being non-existent legislation to determine the technical characteristics and the nutritional quality of such products. Therefore, the aim of this study was to determine the technological and nutritional composition of the “Queijo de Porco” produced in southern Brazil, analyzing the chemical composition, water activity, pH, chloride and residual nitrite content, total and nitrous heme pigments, mineral and collagen content, fatty acids composition and the texture profile analysis. Traditional products from four different regions of Rio Grande do Sul state were used. There were significant differences (P &lt; 0.05) between the brands studied, except for the protein, ash and chlorides contents. The analyzed products show a similar fatty acid content to that reported in the literature for Brazilian pork products. The results obtained indicate that “Queijo de Porco” shows high protein and iron content, with good polyunsaturated/saturated fatty acids ratio and low atherogenic and thrombogenic indexes, which makes it a more healthful product than some pork products produced in Brazil.

Effect of assuming constant tissue scattering on measured tissue oxygenation values during tissue ischemia and vascular reperfusion.

The purpose of this study was to determine the effects of assuming constant tissue scattering properties on tissue oxygenation measurements during a vascular occlusion test (VOT). Twenty-one subjects (21.8 ± 1.9 yr) completed a VOT [1 min baseline (BL), 5 min of tissue ischemia (TI), and 3 min of vascular reperfusion (VR)]. Absolute concentrations of oxygenated heme (oxy-[heme]), deoxygenated heme (deoxy-[heme]), total heme (total [heme), tissue oxygen saturation (StO2), and heme difference [heme]diff) were measured using frequency domain near-infrared spectroscopy (FD-NIRS) while 1) continuously measuring and incorporating tissue scattering ([Formula: see text]) and 2) assuming scattering remained constant. FD-NIRS measured [Formula: see text] increased during TI at 692 nm (P < 0.001) and decreased at 834 nm (P < 0.001). During VR, [Formula: see text] decreased at 692 nm (P < 0.001) and increased at 834 nm (P < 0.001). When assuming constant scattering, oxy-[heme] was significantly less at TIpeak (P < 0.05) while deoxy-[heme] and StO2 were significantly altered at BL, TIpeak, and VRpeak (all P < 0.001). Total [heme] did not change during the VOT. Absolute changes in deoxy-[heme], oxy-[heme], and StO2 in response to TI and VR were significantly exaggerated (all P < 0.001) and the rates of change during TI (slope 1) and VR (slope 2) in deoxy-[heme], oxy-[heme], StO2, and [heme]diff were significantly increased (all P < 0.05) when constant tissue scattering was assumed. These findings demonstrate the need for caution when interpreting NIRS data without continuously measuring tissue optical properties. Further, assuming tissue optical properties remain constant may have important consequences to experimental data and clinical conclusions made using NIRS.NEW & NOTEWORTHY NIRS measurements provide significant experimental and clinical insight. We demonstrate that absolute changes in tissue oxygenation measurements made with NIRS are overestimated and the kinetic responses of NIRS measurements are exaggerated by varying degrees among individuals if tissue scattering characteristics are assumed to remain constant during vascular occlusion tests.

Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-DOPA production

Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host’s genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.

Total plasma heme concentration increases after red blood cell transfusion and predicts mortality in critically ill medical patients.

Relationships between red blood cell (RBC) transfusion, circulating cell-free heme, and clinical outcomes in critically ill transfusion recipients are incompletely understood. The goal of this study was to determine whether total plasma heme increases after RBC transfusion and predicts mortality in critically ill patients. This was a prospective cohort study of 111 consecutive medical intensive care patients requiring RBC transfusion. Cell-free heme was measured in RBC units before transfusion and in the patients' plasma before and after transfusion. Total plasma heme levels increased in response to transfusion, from a median (interquartile range [IQR]) of 35 (26-76) μmol/L to 47 (35-73) μmol/L (p < 0.001). Posttransfusion total plasma heme was higher in nonsurvivors (54 [35-136] μmol/L) versus survivors (44 [31-65] μmol/L, p = 0.03). Posttransfusion total plasma heme predicted hospital mortality (odds ratio [95% confidence interval] per quartile increase in posttransfusion plasma heme, 1.76 [1.17-2.66]; p = 0.007). Posttransfusion total plasma heme was not correlated with RBC unit storage duration and weakly correlated with RBC unit cell-free heme concentration. Total plasma heme concentration increases in critically ill patients after RBC transfusion and is independently associated with mortality. This transfusion-associated increase in total plasma heme is not fully explained by RBC unit storage age or cell-free heme content. Additional studies are warranted to define mechanisms of transfusion-related plasma heme accumulation and test prevention strategies.

The Ribonucleotide Reductase Inhibitor, Didox, Reduces the In Vivo Vascular Inflammation and Oxidative Stress Induced By Acute Hemolysis

Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P<0.01; total plasma heme: 26.3±4.9 and 87.1±18.4 µM, for CON and HEM, respectively, N=≥6, P<0.001). Interestingly, pre-administration of mice with didox slightly, but significantly, decreased plasma Hb and Heme (plasma Hb: 0.43±0.07 and 0.54±0.07 mg/ml, P<0.05; total plasma heme: 62.7.3±9.5 and 71.5±6.7 µM for i.v. and i.p. didox, respectively, N=6, P<0.05). Concomitantly, acute hemolysis significantly augmented leukocyte (WBC) recruitment and extravasation in the cremaster microcirculation (WBC adhesion: 3.07±0.28 and 13.41±1.02 WBC µm-1, P<0.001; WBC extravasation: 0.72±0.16 and 2.93±0.45 WBC (100x50 µm-2) for CON and HEM, respectively, N=≥5, P<0.001). Didox significantly abrogated this effect of hemolysis, decreasing WBC adhesion to 6.12±0.62 and 2.10±0.30 WBC µm-1 and WBC extravasation to 1.32±0.24 and 1.14±0.21 WBC (100x50 µm-2) (for i.v. and i.p. didox, respectively, N≥5, P<0.001). Flow cytometry demonstrated that hemolysis elevated both the generation of reactive oxygen species (ROS) in the granulocytes of mice and their expression of the Mac-1 integrin subunit, CD11b (ROS: 590±58 and 1140±187 MFI, P<0.01; CD11b: 3028±387 and 4534±416 MFI, for CON and HEM, respect. N=3-6, P<0.01). Importantly, the inhibition of vascular inflammation by didox was associated with a significant decrease in both ROS generation and CD11b expression by granulocytes (ROS: 591±53 and 699±120 MFI, P<0.01; CD11b 2399±126 and 2379±207 MFI, for i.v. and i.p. didox, respectively. N=3-6, P<0.01). Conclusion: Didox, when administrated both intravascularly and intraperitoneally in mice, can inhibit the significant elevations in leukocyte integrin expression and cellular recruitment in the microcirculation that are induced by acute hemolysis. This improvement in vascular inflammation is accompanied by the prevention of oxidative stress in the leukocytes. While didox was found to slightly reduce hemolysis in response to osmotic shock, an interesting observation in itself, it is probable that the beneficial effects of this molecule on vascular inflammation are mediated largely by its effect on oxidative stress parameters. As such, this molecule may provide a novel therapeutic approach to reduce the oxidative stress and vascular inflammation caused by hemolytic events. This includes potential clinical use to treat sickle cell disease and other disorders in which hemolysis is a contributing factor to the pathophysiology. DisclosuresElford:Molecules for Health, Inc.: Equity Ownership, Patents & Royalties: Didox.

Acute Hemolysis Induces Pro-Angiogenic Molecule Production and Neovascularization In Vivo

Background: The intravascular hemolysis associated with hemolytic disorders, such as sickle cell anemia, results in the release of cell-free hemoglobin (Hb) and heme in the circulation, in turn, inducing inflammatory processes, vascular damage and endothelial activation. Angiogenesis, or the formation of new blood vessels, involves the proliferation, migration, and reorganization of endothelial cells in response to diverse physiological or pathological stimuli. Although angiogenesis is important for tissue growth and regeneration, uncontrolled angiogenesis can result in the accumulation of inflammatory cells, together with fibrosis and ischemia, and could play a role in some of the complications of hemolytic disorders. Aim: The aim of this study was to characterize angiogenic and inflammatory responses to the hemolytic process using an experimental in vivo model of acute hemolysis. Methods: C57BL/6J male mice were submitted, or not, to an osmotic hemolytic stimulus by intravascular injection of 150μL of sterile water (HEM group) or saline (CON group). After 1, 24 and 120 h, blood was collected by cardiac puncture for cell counts and plasma Hb and total heme quantification (colorimetric assays). Hemopexin, haptoglobin, inflammatory cytokines (Interleukin [IL]-6, IL-1β and IL-10) and angiogenic factors (Angiopoietin-2, Fibroblast growth factor [FGF]-basic, Platelet-derived growth factor [PDGF]-AA, PDGF-BB, Trombospondin-4, Vascular endothelial growth factor [VEGF], VEGFR2) were quantified in plasma by immunoassay. To evaluate in vivo neovascularization, a Matrigel®/ heparin mixture was injected subcutaneously into the dorsal region of the mice, two days before the administration of the hemolytic stimulus. After five more days, the Matrigel® plugs were removed, photographed and neovascularization quantified by colorimetric measurement of Hb in the plug. Results: At 1 h after the acute hemolytic stimulus, significant increases were observed in plasma Hb and heme (0.1±0.02 vs 0.2±0.03 g/L Hb, p<0.001; 32.9±1.9 vs 50.04±4.6 µM heme, p<0.01, for CON and HEM [1h], respect., n=5), indicative of the induction of hemolysis. Haptoglobin levels were almost completely depleted at 1 h after hemolysis, but recovered and were even higher at 24 h (14.2±2.9 vs 1.1±0.3 and 34.6±2.1 ng/mL for CON, HEM [1h] and HEM [24h], respect., p<0.01, n=5), before normalizing at 120 h. In contrast, circulating levels of hemopexin were not altered at any time post hemolysis (data not shown, p>0.05). Hemolysis also elevated the white blood cell count (2.2±0.2 vs 3.5±0.3 103/µL for CON and HEM [1h], respect., p<0.05, n=5) and plasma IL-10 (4.8±0.6 vs 12.4±1.7 pg/mL for CON and HEM [1h] respect., p<0.001, n=10 and n=5) within 1h, suggesting that systemic inflammation accompanied this hemolysis. The red blood cell count did not change in the HEM group at any of the time points, nor did plasma IL-1β or IL-6 levels (p>0.05). A balance of angiogenic mediators, including growth factors and inflammatory cytokines, regulates angiogenesis; at 1 h after hemolysis, plasma levels of angiopoietin-2 were decreased (2.75±0.1 vs 2.1±0.15 ng/mL for CON and HEM [1h], respect., p<0.05, n=5), while pro-angiogenic VEFG and trombospondin-4 were significantly increased (163.1±8.9 vs 233.3±15.8 pg/mL and 148.6±4.1 vs 169.4±7.1 ng/mL for CON and HEM [1h], respect., p<0.05, n=5), by 24 h levels of angiogenic markers were normalized. In association with the alterations in the molecular angiogenic profile of mice after hemolysis, the formation of new blood vessels in dorsal Matrigel® plugs was significantly elevated during the 5 days following the hemolytic stimulus, as quantified by plug Hb content (2.0±0.3 vs 3.0±0.04 µg/mL for CON and HEM mice, respect., p<0.05, n=10, 11). Conclusions: Acute intravascular hemolysis was associated with rapid alterations in circulating angiogenic and inflammatory markers in mice. In association with this pro-angiogenic profile, in vivo neovascularization was accelerated in animals following hemolysis. These data suggest that hemolysis may be a significant stimulus for angiogenic processes, which in turn may contribute to some of the clinical complications of hemolytic diseases, including pulmonary hypertension, stroke and leg ulcers. Furthermore, the angiogenic process may represent a target for the development of therapeutic strategies in disorders characterized by hemolysis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 16728: Cigarette Smoke Impairs the Anti-Oxidative Response by Reducing Heme Levels and Inducing Bach1

Introduction: Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. Hypothesis: We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods: Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) (3 mg/ml nicotine) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Llevels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using the caspase 3/7 assay. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results: Exposure to CSE induced the expression of BACH1 (1.90±0.18; p&lt;0.05) and reduced total heme levels (0.30±0.04; p&lt;0.001) in HUVEC. It also reduced miR-125b expression in HUVEC (0.39±0.09; p&lt;0.001) and umbilical veins (0.72±0.12; p&lt;0.05), whereas the expression of miR-125b target AhR repressor ( AHRR ) increased (14.93±4.08; p&lt;0.05). Inhibiting BACH1 rescued miR-125b expression (1.02±0.45; p&gt;0.05) and reduced AHRR expression (0.66±0.12; P&lt;0.05). Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a 2-fold reduction in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 (1.33±0.05; p&lt;0.01) and an increase in expression of anti-oxidative HMOX1 (18.18±1.8; p&lt;0.001) and NQO1 (16.12±1.99; p&lt;0.001). CSE-induced apoptosis (1.23±0.04; p&lt;0.001) was reversible by pre-conditioning with 1 mM N-acetyl cysteine (0.99±0.02; p&gt;0.05) or overexpression of miR-125b (0.99±0.07; p&gt;0.05). Conclusions: These results show that cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b in the human endothelium. Reduced heme levels could be involved in the upregulation of Bach1 activity.

1 - Cigarette smoke impairs the anti-oxidative response by reducing heme levels and inducing Bach1

Introduction Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Levels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using caspase 3/7 activity. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results Exposure to CSE induced the expression of BACH1 and reduced total heme levels in HUVEC. It also reduced miR-125b expression in HUVEC and umbilical veins, whereas the expression of miR-125b target AhR repressor (AHRR) increased. Inhibiting BACH1 increased miR-125b expression and reduced AHRR expression. Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a decrease in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 and an increase in expression of HMOX1 and NQO1. CSE-induced apoptosis was reversible by preconditioning with N-acetyl cysteine or overexpression of miR-125b. Conclusions cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b. Reduced heme levels could be involved in the upregulation of Bach1 activity.

Metabolic engineering of Escherichia coli for secretory production of free haem

Haem has widespread applications in healthcare and food supplement industries. Escherichia coli has previously been engineered to produce a small amount of haem intracellularly through the C4 pathway, requiring extraction for applications. Here we report secretory production of free haem by engineered E. coli strains, using the C5 pathway and the optimized downstream pathway for haem biosynthesis. Furthermore, knocking out ldhA, pta and also yfeX—encoding a putative haem-degrading enzyme—results in 7.88 mg l−1 of total haem with 1.26 mg l−1 of extracellular haem in flask cultivation. Fed-batch fermentations of the engineered strain overexpressing a haem exporter CcmABC from glucose only and glucose supplemented with l-glutamate secrete 73.4 and 151.4 mg l−1 of haem, respectively, which are 63.5% of 115.5 mg l−1 and 63.3% of 239.2 mg l−1 of total haem produced. The engineered E. coli strain reported here will be useful for microbial production of free haem. Microbial production of haem for applications in healthcare and food supplement industry requires high-performing strains. Here, Lee and co-workers report secretory production of free haem by metabolically engineered Escherichia coli strains to produce up to 239 mg l−1 total haem.

Heme bioavailability and signaling in response to stress in yeast cells

Protoheme (hereafter referred to as heme) is an essential cellular cofactor and signaling molecule that is also potentially cytotoxic. To mitigate heme toxicity, heme synthesis and degradation are tightly coupled to heme utilization in order to limit the intracellular concentration of "free" heme. Such a model, however, would suggest that a readily accessible steady-state, bioavailable labile heme (LH) pool is not required for supporting heme-dependent processes. Using the yeast Saccharomyces cerevisiae as a model and fluorescent heme sensors, site-specific heme chelators, and molecular genetic approaches, we found here that 1) yeast cells preferentially use LH in heme-depleted conditions; 2) sequestration of cytosolic LH suppresses heme signaling; and 3) lead (Pb2+) stress contributes to a decrease in total heme, but an increase in LH, which correlates with increased heme signaling. We also observed that the proteasome is involved in the regulation of the LH pool and that loss of proteasomal activity sensitizes cells to Pb2+ effects on heme homeostasis. Overall, these findings suggest an important role for LH in supporting heme-dependent functions in yeast physiology.

Depletion of haptoglobin and hemopexin promote hemoglobin-mediated lipoprotein oxidation in sickle cell disease.

Intravascular sickling and lysis of red blood cells, a hallmark feature of sickle cell disease (SCD), releases hemoglobin (Hb) into the circulation. Increased cell-free Hb has been linked to vasculopathy and in vitro lipid oxidation. Scavenger plasma proteins haptoglobin (Hp) and hemopexin (Hpx) can attenuate cell-free Hb and total plasma heme lipid-oxidative capacity but are depleted in SCD. Here, we isolated lipids from BERK-SS mice, guinea pigs (GP) infused with heme-albumin, and patients with SCD undergoing regular exchange transfusion therapy and evaluated the level of lipid oxidation. Malondialdehyde formation, an end product of lipid peroxidation, was increased in BERK-SS mice, purified lipid fractions of the heme-albumin infused GP, and patients with SCD compared with controls. In humans, the extent of lipid oxidation was associated with the absence of Hp as well as decreased Hpx in plasma samples. Postmortem pulmonary tissue obtained from patients with SCD demonstrated oxidized LDL deposition in the pulmonary artery. The relationship between no Hp and low Hpx levels with greater LDL and HDL oxidation demonstrates the loss of protection against cell-free Hb and total plasma heme-mediated lipid oxidation and tissue injury in SCD. Strategies to protect against plasma lipid oxidation by cell-free Hb and total plasma heme (e.g., therapeutic Hp and Hpx replacement) may diminish the deleterious effects of cell-free Hb and total plasma heme toward the vascular system in SCD.

Abstract 2409: The effect of heme on mitochondrial biogenesis and function of NSCLC cells

Abstract Heme is a central molecule for mitochondrial function and for many processes involved in oxygen utilization. Heme serves as a prosthetic group or a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme also directly regulates the synthesis, translocation, and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiologic studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. To assess the status of heme metabolism in lung cancer cells, we performed a series of experiments in non-small cell lung cancer (NSCLC) cell lines and compared the results with an immortalized normal lung cell line, HBEC30KT. We observed significant increase in rates of heme synthesis and heme uptake in NSCLC cells compared to HBEC30KT. Previous studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme as well as hemoproteins in NSCLCs. NSCLCs were also found to exhibit significantly high expressions of proteins involved in mitochondrial biogenesis such as SIRT-1, PGC1α, NRF-1, NRF-2, and TFAM. Expression levels of these proteins correlate with oxygen consumption and total heme content in NSCLCs, suggesting a possibility of role of heme in mitochondrial biogenesis. Immunofluoresence studies showed that heme depletion and add-back significantly affected localization and expression levels of these proteins. Studies are currently under way to determine how heme depletion affects the interactions of various transcription factors involved in mitochondrial biogenesis and function. Citation Format: Sagar Shashikant Sohoni, Chantal Vidal, Li Zhang. The effect of heme on mitochondrial biogenesis and function of NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2409.

Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period

The periparturient period is the most critical period during the lactation cycle of dairy cows and is characterized by increased oxidative stress status. The objective of this experiment was to evaluate the effect of supplementing rumen-protected methionine on nuclear factor erythroid 2-like 2 (NFE2L2, formerly NRF2) protein and target gene expression in the mammary gland during the early postpartal period. Multiparous Holstein cows were used in a block design experiment with 30 cows per treatment. Treatments consisting of a basal control diet (control) or the basal diet plus rumen-protected methionine (methionine) were fed from d -28 to 60 relative to parturition. Mammary tissue biopsies were harvested on d 21 postpartum from 5 cows per treatment. Compared with control, methionine increased dry matter intake, milk yield, and milk protein content. Among plasma parameters measured, methionine led to greater methionine and lower reactive oxygen metabolites. Compared with control, methionine supply resulted in greater mRNA abundance of the NFE2L2 target genes glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), malic enzyme 1 (ME1), ferrochelatase (FECH), ferritin heavy chain 1 (FTH1), and NAD(P) H quinone dehydrogenase 1 (NQO1) in the mammary tissue. In addition, methionine upregulated the mRNA abundance of NFE2L2, NFKB1, MAPK14 and downregulated KEAP1. The ratio of phosphorylated NFE2L2 to total NFE2L2 protein, and total heme oxygenase 1 (HMOX1) protein were markedly greater in response to methionine supply. In contrast, total protein abundance of Kelch-like ECH-associated protein 1 (KEAP1), which sequesters NFE2L2 in the cytosol and reduces its activity, was lower with methionine. Besides the consistent positive effect of methionine supply on systemic inflammation and oxidative stress status, the present data indicate a positive effect also on antioxidant mechanisms within the mammary gland, which are regulated, at least in part, via phosphorylation of NFE2L2 and its target genes. The exact mechanisms for these responses merit further study.

Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease.

Butyric acid (BA) is produced by periodontopathic bacterial pathogens and contributes to periodontal disease (PD) induction. Moreover, PD has been associated with detrimental effects which subsequently may lead to systemic disease (SD) development affecting certain organs. Surprisingly, the potential systemic manifestations and organ-localized effects of BA have never been elucidated. Here, we simulated BA-based oral infection among young (20-week-old) rats and isolated blood cytosol to determine BA effects on stress network-related signals [total heme, hydrogen peroxide (H2O2), catalase (CAT), glutathione reductase (GR), free fatty acid (FFA), NADP/NADPH], inflammation-associated signals [caspases (CASP12 and CASP1), IL-1β, TNF-α, metallomatrix proteinase-9 (MMP-9), and toll-like receptor-2 (TLR2)], and neurological blood biomarkers [presenilin (PS1 and PS2) and amyloid precursor protein (APP)]. Similarly, we extracted the brain from both control and BA-treated rats, isolated the major regions (hippocampus, pineal gland, hypothalamus, cerebrum, and cerebellum), and, subsequently, measured stress network-related signals [oxidative stress: total heme, NADPH, H2O2, GR, and FFA; ER stress: GADD153, calcium, CASP1, and CASP3] and a brain neurodegenerative biomarker (Tau). In the blood, we found that BA was no longer detectable. Nevertheless, oxidative stress and inflammation were induced. Interestingly, amounts of representative inflammatory signals (CASP12, CASP1, IL-1β, and TNF-α) decreased while MMP-9 levels increased which we believe would suggest that inflammation was MMP-9-modulated and would serve as an alternative inflammatory mechanism. Similarly, TLR2 activity was increased which would insinuate that neurological blood biomarkers (APP, PS1, and PS2) were likewise affected. In the brain, BA was not detected, however, we found that both oxidative and ER stresses were likewise altered in all brain regions. Interestingly, tau protein amounts were significantly affected in the cerebellar and hippocampal regions which coincidentally are the major brain regions affected in several neurological disorders. Taken together, we propose that gingival BA can potentially cause systemic inflammation ascribable to prolonged systemic manifestations in the blood and localized detrimental effects within the brain organ.

Near-Infrared Spectroscopy Derived Total Heme vs. Assay Derived Total Heme

Near-Infrared Spectroscopy Derived Total Heme vs. Assay Derived Total Heme

Bleeding of Common Carp (Cyprinus carpio) Improves Sensory Quality of Fillets and Slows Oxidative and Microbiological Changes During Refrigerated Aerobic Storage.

SUMMARYCommon carp (Cyprinus carpio) aquaculture is one of the most important and rapidly growing productions around the world. However, for consumers, carp is often not acceptable due to its distinctive colour and odour. In this study, we investigated the effects of bleeding of common carp on fillet quality. The obtained results show that carp bleeding by cutting the gill arches is an effective way of reducing the total haem content, which here decreased from (9.6±1.6) in unbled carp to (2.34±0.8) μmol/kg of haemoglobin in bled carp. Furthermore, fillets from bled carp showed reduced formation of primary and secondary lipid oxidation products and growth of microorganisms during 12 days of refrigerated aerobic storage. On the last day of storage, the amount of lipid hydroperoxides decreased from (88.9±4.2) in unbled to (62.1±2.9) μmol/kg of cumene hydroperoxide in bled carp, TBARS decreased from (4.2±0.5) in unbled to (2.6±0.4) μmol/kg of malondialdehyde in bled carp, mesophilic and psychrotrophic bacteria count decreased from (6.4±0.1) and (6.2±0.3) log CFU/g in unbled to (4.0±0.2) and (4.2±0.2) log CFU/g in bled carp, respectively. These raw bled fillets showed increased lightness L*, and reduced redness a* and yellowness b* compared to unbled fillets. Sensory analysis showed improved colour, odour and overall acceptability of bled raw fillets. Overall, bleeding improves the quality of carp fillets. Thus, inclusion of bleeding into processing of carp fillets has the potential to improve their acceptance by consumers and prolong their shelf-life.

Effect of ultrasound on the physicochemical and microbiological characteristics of Italian salami

Italian salami were sonicated in different times (0, 3, 6 and 9min) using ultrasound bath (US, 25kHz). The effect of sonication on microbial growth (lactic acid bacteria and Micrococcaceae), lipid and protein oxidation, total heme pigments (THP), non heme iron (NHI) and metmyoglobin (MMb) was investigated during processing (0, 2, 15, and 28days) and storage (1, 30, and 120days). US enhanced growth of microorganisms (P<0.05), mainly for the treatment 9min of sonication. The lipid (peroxide value and TBARS) and protein (thiol group) oxidative reactions were accelerated by US (P<0.05) and they should be considered to maintain Italian salami quality. Sonication contributed to maintenance of THP (P<0.05), especially during storage. MMb pigment was not affected by sonication (P>0.05). This study presented some features of US application that could be explored in the manufacture of Italian salami.

Haem is associated with thrombosis in neonates and infants undergoing cardiac surgery for congenital heart disease.

Haem levels are associated with thrombosis in a variety of diseases, as well as being a contributing cause of thrombotic events in animal models. We retrospectively analyzed samples from 39 children who underwent cardiac surgery with cardiopulmonary bypass, including 15 children who developed a postoperative thrombosis and 24 controls. Patients who developed thrombosis postoperatively had statistically significant higher average haem levels over time (presurgery to 12 h postsurgery) compared to patients who did not develop thrombosis. Higher cell-free total haem levels are associated with a higher risk of thrombosis in a paediatric cardiac surgical cohort.