Photomedicine based on heme-derived compounds.

Photoimaging and phototherapy have become major platforms for the diagnosis and treatment of various health complications. These applications require a photosensitizer (PS) that is capable of absorbing light from a source and converting it into other energy forms for detection and therapy. While synthetic inorganic materials such as quantum dots and gold nanorods have been widely explored for their medical diagnosis and photodynamic (PDT) and photothermal (PTT) therapy capabilities, translation of these technologies has lagged, primarily owing to potential cytotoxicity and immunogenicity issues. Of the various photoreactive molecules, the naturally occurring endogenous compound heme, a constituent of red blood cells, and its derivatives, porphyrin, biliverdin and bilirubin, have shown immense potential as noteworthy candidates for clinically translatable photoreactive agents, as evidenced by previous reports. While porphyrin-based photomedicines have attracted significant attention and are well documented, research on photomedicines based on two other heme-derived compounds, biliverdin and bilirubin, has been relatively lacking. In this review, we summarize the unique photoproperties of heme-derived compounds and outline recent efforts to use them in biomedical imaging and phototherapy applications.

Intravenous Heme Arginate Induces HO-1 (Heme Oxygenase-1) in the Human Heart.

Objective- HO-1 (heme oxygenase-1) induction may prevent or reduce ischemia-reperfusion injury. We previously evaluated its in vivo induction after a single systemic administration of heme arginate in peripheral blood mononuclear cells. The current trial was designed to assess the pharmacological tissue induction of HO-1 in the human heart with heme arginate in vivo. Approach and Results- Patients planned for conventional aortic valve replacement received placebo (n=8), 1 mg/kg (n=7) or 3 mg/kg (n=9) heme arginate infused intravenously 24 hours before surgery. A biopsy of the right ventricle was performed directly before aortic cross-clamping and after cross-clamp release. In addition, the right atrial appendage was partially removed for analysis. HO-1 protein and mRNA concentrations were measured in tissue samples and in peripheral blood mononuclear cells before to and up to 72 hours after surgery. No study medication-related adverse events occurred. A strong, dose-dependent effect on myocardial HO-1 mRNA levels was observed (right ventricle: 7.9±5.0 versus 88.6±49.1 versus 203.6±148.7; P=0.002 and right atrium: 10.8±8.8 versus 229.8±173.1 versus 392.7±195.7; P=0.001). This was paralleled by a profound increase of HO-1 protein concentration in atrial tissue (8401±3889 versus 28 585±10 692 versus 29 022±8583; P<0.001). Surgery and heme arginate infusion significantly increased HO-1 mRNA concentration in peripheral blood mononuclear cells ( P<0.001). HO-1 induction led to a significant increase of postoperative carboxyhemoglobin (1.7% versus 1.4%; P=0.041). No effect on plasma HO-1 protein levels could be detected. Conclusions- Myocardial HO-1 mRNA and protein can be dose-dependently induced by heme arginate. Protective effects of this therapeutic strategy should be evaluated in upcoming clinical trials. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT02314780.

[Present-day possibilities of non-invasive control over microcirculation and metabolism in man]. / Sovremennye vozmozhnosti neinvazivnogo kontrolia mikrotsirkuliatsii i obmena veshchestv u cheloveka.

The main function of the microcirculatory bed consists in maintaining tissue homeostasis at an optimal level irrespective of the effect of various external and internal factors. Of all types of metabolism (diffusive, filtration-reabsorption and vesicular), directly dependent on the haemodynamic parameters is filtration-reabsorption metabolism which provides exchange of water, low-molecular-weight and water-soluble substances at the opposite to the heart «pole¼ of the cardiovascular system. The present study was aimed at testing a hypothesis that activation of metabolic processes in man would be accompanied by alterations in haemodynamic parameters which may be registered by means of modern non-invasive methods of examination, i. e., laser Doppler flowmetry (LDF) and computer-assisted capillaroscopy (CCS). We used actovegin as an activator of metabolic processes. The study included acute pharmacological testing in apparently healthy volunteers (n=28), a course of taking actovegin in patients with cognitive dysfunctions on the background of arterial hypertension (n=60) and in patients with chronic ischaemia of the lower limbs (n=80). The obtained findings of LDF and CCS demonstrated that the known metabolic effects of actovegin (improved utilization of oxygen and glucose by tissues) were accompanied by an increase in the number of functioning capillaries, increased velocity of capillary blood flow with a decrease in the degree of hydration of the interstitial space, thus reducing the «blood-cell¼ distance for nutrients and products of tissue metabolism. Improvement of capillary blood flow was determined by a decrease in the tonicity of the capillary sphincters, thus leading to reduced arteriolar-venular shunting of blood with predominant supply to the capillary bed, improved NO-mediated regulation of the value of the lumen of the precapillary arterioles by the microvascular endothelium, improved reaction of resistant microvessels to various dilatation stimuli. The obtained results make it possible to draw a conclusion that modern non-invasive methods of study of human microcirculation (LDF and CCS) are informative not only for assessment of the functional state of the microcirculatory bed of the skin but make it possible to evaluate efficacy of the filtration-reabsorption mechanism of metabolism.

[Correction of small bowel function as a new direction for treating patients with metabolic syndrome]. / Korrektsiia narushenii funktsional'nogo sostoianiia tonkoi kishki kak novoe napravlenie lecheniia bol'nykh s metabolicheskim sindromom.

AIM: To provide a rationale for and to evaluate the therapeutic efficiency of the combined use of pancreatic enzymes and actovegin in the combination therapy of patients with metabolic syndrome (MS) on the basis of comprehensive clinical and functional studies of the small bowel (SB). SUBJECTS AND METHODS: In the course of treatment, 120 patients with MS (verified using the diagnostic criteria elaborated by the All-Russian Research Society of Cardiology (2009)) underwent a comprehensive study of SB function: an isolated study of resorptive processes; evaluation of parietal and cavitary digestion, motor-evacuation function. The peripheral blood levels of gastrin, insulin, cortisol, thyroxine and thyrotropin were determined. RESULTS: The combined use of pancreatic enzymes and actovegin has a positive impact on the clinical and functional state of SB, which was manifested as restoration of its hydrolysis and absorption, as well as motor-evacuation function in the patients with MS. The treatment resulted in reductions in the levels of triglycerides from 2.85±0.34 to 1.53±0.18 mmol/l (p<0.01), total cholesterol from 6.08±0.16 to 5.19±0.21 mmol/l (p<0.05), and atherogenic factor from 5.21±0.28 to 2.93±0.34 (p<0.05). Posttreatment HOMA-IR decreased from 4.22±0.8 to 2.12±0.8. There were no substantial changes in insulin levels and insulin resistance index in the patients on standard therapy. CONCLUSION: The combined use of pancreatic enzymes and actovegin is pathogenetically sound in correcting SB dysfunctions and may be one of the most effective directions for the treatment of patients with MS.

Physiological stress-induced corticosterone increases heme uptake via KLF4-HCP1 signaling pathway in hippocampus neurons.

Iron overload has attracted much attention because of its adverse effect in increasing the risk of developing several neurodegenerative disorders. Under various pathologic conditions, a lot of heme are released. The aggregation of heme is more neurotoxic than that of iron released from the heme breakdown. Our previous studies demonstrated that psychological stress (PS) is a risk factor of cerebral iron metabolism disorders, thus causing iron accumulation in rat brains. In the present study, we found PS could increase heme uptake via heme carrier protein 1 (HCP1) in rat brains. We demonstrated that Glucocorticoid (GC), which is largely secreted under stress, could up-regulate HCP1 expression, thus promoting heme uptake in neurons. We also ascertained that HCP1 expression can be induced by GC through a transcription factor, Krüppel-like factor 4 (KLF4). These results may gain new insights into the etiology of heme uptake and iron accumulation in PS rats, and find new therapeutic targets of iron accumulation in Parkinson's disease or Alzheimer's disease.

Prebiotics increase heme iron bioavailability and do not affect non-heme iron bioavailability in humans.

The aim of this study was to establish the effect of a prebiotic mix on heme and non-heme iron (Fe) bioavailability in humans. To this purpose, twenty-four healthy women were randomized into one of two study groups. One group ate one yogurt per day for 12 days with a prebiotic mix (prebiotic group) and the other group received the same yogurt but without the prebiotic mix (control group). Before and after the intake period, the subjects participated in Fe absorption studies. These studies used 55Fe and 59Fe radioactive isotopes as markers of heme Fe and non-heme Fe, respectively, and Fe absorption was measured by the incorporation of radioactive Fe into erythrocytes. The results showed that there were no significant differences in heme and non-heme Fe bioavailability in the control group. Heme Fe bioavailability of the prebiotic group increased significantly by 56% post-prebiotic intake. There were no significant differences in non-heme Fe bioavailability in this group. We concluded that daily consumption of a prebiotic mix increases heme Fe bioavailability and does not affect non-heme iron bioavailability.

Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat.

Actovegin, a non-prohibited drug increases oxidative capacity in human skeletal muscle.

Actovegin, a deproteinized haemodialysate of calf blood, is suggested to have ergogenic properties, but this potential effect has never been investigated in human skeletal muscle. To investigate this purported ergogenic effect, we measured the mitochondrial respiratory capacity in permeabilized human skeletal muscle fibres acutely exposed to Actovegin in a low and in a high dose. We found that Actovegin, in the presence of complex I-linked substrates increased the oxidative phosphorylation (OXPHOS) capacity significantly in a concentration-dependent manner (19 ± 3, 31 ± 4 and 45 ± 4 pmol/mg/s). Maximal OXPHOS capacity with complex I and II-linked substrate was increased when the fibres were exposed to the high dose of Actovegin (62 ± 6 and 77 ± 6 pmol/mg/s) (p < .05). The respiratory capacity of the electron transfer system as well as Vmax and Km were also increased in a concentration-dependent manner after Actovegin exposure (70 ± 6, 79 ± 6 and 88 ± 7 pmol/mg/s; 13 ± 2, 25 ± 3 and 37 ± 4 pmol/mg/s; 0.08 ± 0.02, 0.21 ± 0.03 and 0.36 ± 0.03 mM, respectively) (p < .05). In summary, we report for the first time that Actovegin has a marked effect on mitochondrial oxidative function in human skeletal muscle. Mitochondrial adaptations like this are also seen after a training program in human subjects. Whether this improvement translates into an ergogenic effect in athletes and thus reiterates the need to include Actovegin on the World Anti-Doping Agency's active list remains to be investigated.

Heme iron intake and acute myocardial infarction: a prospective study of men.

BACKGROUND: Epidemiologic studies of heme iron and non-heme iron intake in relation to risk of acute myocardial infarction (AMI) are lacking. Therefore, we examine the associations between heme iron and non-heme iron intake and fatal and nonfatal AMI in men. Moreover, we investigated whether the associations were modified by intake of minerals (calcium, magnesium, and zinc) that decreases iron absorption. METHODS: The population-based prospective cohort of Swedish Men (COSM) included 36882 men, aged 45-79 years, who completed a self-administered questionnaire on diet and had no history of coronary heart disease, stroke, diabetes, or cancer at baseline. RESULTS: During an 11.7 year follow-up, 678 fatal and 2593 nonfatal AMI events were registered. The hazard ratio (HR) of fatal AMI among men in the highest compared with the lowest quintile of heme iron intake was 1.51 (95%CI: 1.07-2.13, P-trend=0.02). The association was confined to men with a low intake of minerals that can decrease iron absorption. Among men with combined intakes of calcium, magnesium, and zinc below the medians, the HR of fatal AMI was 2.89 (95%CI: 1.43-5.82) for the highest vs. the lowest quintile of heme iron intake. There was no association between heme iron intake and nonfatal AMI, or between non-heme iron intake and fatal or nonfatal AMI. CONCLUSIONS: Findings from this prospective study indicate that a high heme iron intake, particularly with simultaneous low intake of minerals that can decrease iron absorption, may increase the risk of fatal AMI.

The P. aeruginosa heme binding protein PhuS is a heme oxygenase titratable regulator of heme uptake.

The Pseudomonas aeruginosa heme utilization (Phu) system encodes several proteins involved in the acquisition of heme as an iron source. Once internalized, heme is degraded by the iron-regulated heme oxygenase, HemO to biliverdin (BV) IXδ and ß. In vitro studies have shown holo-PhuS transfers heme to the iron-regulated HemO. This protein-protein interaction is specific for HemO as PhuS does not interact with the α-regioselective heme oxygenase, BphO. Bacterial genetics and isotopic labeling ((13)C-heme) studies confirmed extracellular heme is converted to (13)C-BVIX δ and ß through the catalytic action of HemO. In an effort to further understand the role of PhuS, similar studies were performed on the P. aeruginosa PAO1 ΔphuS and ΔphuS/ΔhemO strains. In contrast to wild-type strain, the absence of PhuS results in extracellular heme uptake and degradation via the catalytic action of HemO and BphO. At low heme concentrations, loss of PhuS leads to inefficient extracellular heme uptake supported by the fact the mRNA levels of PhuR, HemO, and BphO remain elevated when compared to the wild-type PAO1. On increasing extracellular heme concentrations, the elevated levels of PhuR, HemO, and BphO allow "leaky uptake" and degradation of heme via HemO and BphO. Similarly, in the ΔphuS/ΔhemO strain, the higher heme concentrations combined with elevated levels of PhuR and BphO leads to nonspecific heme uptake and degradation by BphO. Thus we propose heme flux into the cell is driven by the catalytic action of HemO with PhuS acting as a "control valve" to regulate extracellular heme flux.

Heme iron-based dietary intervention for improvement of iron status in young women.

OBJECTIVE: Conventional iron deficiency treatment with pharmacologic iron doses often causes side effects. Heme iron has high bioavailability and a low capacity to cause gastrointestinal side effects. This study investigated the possibility of using heme iron in the form of blood-based crisp bread as a diet-based treatment program to improve the iron status of women of reproductive age. METHODS: In a 12-wk intervention study, 77 women (mean age 24 y) were assigned to one of four groups: blood-based crisp bread (35 mg of iron [Fe], 27 mg of which was heme Fe), iron supplementation consisting of 35 mg of non-heme iron/day (Fe35), iron supplementation consisting of 60 mg of non-heme iron/day (Fe60), and controls (iron-free tablets). RESULTS: Body iron increased significantly in the crisp bread group by a median of 2.7 mg/kg (interquartile range 3.1, n = 18), in the Fe35 group by 2.7 mg/kg (interquartile range 2.8, n = 11), and in the Fe60 group by 4.1 mg/kg (interquartile range 3.6, n = 13), whereas no change was observed in the control group. No statistically significant difference in iron status increase was observed between the crisp bread group compared with the two iron-supplemented groups. CONCLUSION: Dietary-based treatment containing heme iron has few side effects and can be used efficiently to improve the iron status of women of reproductive age.

A heme degradation enzyme, HutZ, from Vibrio cholerae.

HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.

Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake.

The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 µM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.

Absorption of iron from ferritin is independent of heme iron and ferrous salts in women and rat intestinal segments.

Ferritin iron from food is readily bioavailable to humans and has the potential for treating iron deficiency. Whether ferritin iron absorption is mechanistically different from iron absorption from small iron complexes/salts remains controversial. Here, we studied iron absorption (RBC (59)Fe) from radiolabeled ferritin iron (0.5 mg) in healthy women with or without non-ferritin iron competitors, ferrous sulfate, or hemoglobin. A 9-fold excess of non-ferritin iron competitor had no significant effect on ferritin iron absorption. Larger amounts of iron (50 mg and a 99-fold excess of either competitor) inhibited iron absorption. To measure transport rates of iron that was absorbed inside ferritin, rat intestinal segments ex vivo were perfused with radiolabeled ferritin and compared to perfusion with ferric nitrilotriacetic (Fe-NTA), a well-studied form of chelated iron. Intestinal transport of iron absorbed inside exogenous ferritin was 14.8% of the rate measured for iron absorbed from chelated iron. In the steady state, endogenous enterocyte ferritin contained >90% of the iron absorbed from Fe-NTA or ferritin. We found that ferritin is a slow release source of iron, readily available to humans or animals, based on RBC iron incorporation. Ferritin iron is absorbed by a different mechanism than iron salts/chelates or heme iron. Recognition of a second, nonheme iron absorption process, ferritin endocytosis, emphasizes the need for more mechanistic studies on ferritin iron absorption and highlights the potential of ferritin present in foods such as legumes to contribute to solutions for global iron deficiency.

Calcium does not inhibit the absorption of 5 milligrams of nonheme or heme iron at doses less than 800 milligrams in nonpregnant women.

Calcium is the only known component in the diet that may affect absorption of both nonheme and heme iron. However, the evidence for a calcium effect on iron absorption mainly comes from studies that did not isolate the effect of calcium from that of other dietary components, because it was detected in single-meal studies. Our objective was to establish potential effects of calcium on absorption of nonheme and heme iron and the dose response for this effect in the absence of a meal. Fifty-four healthy, nonpregnant women were selected to participate in 4 iron absorption studies using iron radioactive tracers. We evaluated the effects of calcium doses between 200 and 1500 mg on absorption of 5 mg nonheme iron (as ferrous sulfate). We also evaluated the effects of calcium doses between 200 and 800 mg on absorption of 5 mg heme iron [as concentrated RBC (CRBC)]. Calcium was administered as calcium chloride in all studies and minerals were ingested on an empty stomach. Calcium doses ≥1000 mg diminished nonheme iron absorption by an average of 49.6%. A calcium dose of 800 mg diminished absorption of 5 mg heme iron by 37.7%. In conclusion, we demonstrated an isolated effect of calcium (as chloride) on absorption of 5 mg of iron provided as nonheme (as sulfate) and heme (as CRBC) iron. This effect was observed at doses higher than previously reported from single-meal studies, starting at ~800 mg of calcium.

Actovegin®: a biological drug for more than 5 decades.

Actovegin(®) is a biological drug manufactured from a natural source: it is a calf blood hemodialysate. Its therapeutic benefits stem from a variety of pharmacodynamic actions that can be summarized to a common goal, i.e. the enhancement of cellular metabolism; this results from an insulin-like activity mediated by Inositol-phospho-oligosaccharides. Actovegin(®) results in beneficial effects in several pathophysiological clinical settings including malfunction of the blood circulation and trophic disturbances in the brain, impairment of peripheral blood circulation and associated diseases, dermal transplants and acute and chronic wounds. Here, we give an overview of the pharmacodynamic actions of calf-blood hemidialysate and its beneficial effects in a variety of clinical settings.

Effect of dietary protein on heme iron uptake by Caco-2 cells.

OBJECTIVE: To study heme iron bioavailability and the role of dietary protein (animal and vegetable) on iron uptake using an in vitro model (Caco-2 cell line). METHODS: Caco-2 cells were seeded in bicameral chambers with different animal (beef, chicken or fish) or vegetable (peas, lentils, and soybeans) proteins or with pure animal (collagen and casein) or vegetable (gliadin, zein, and glutein) protein extracts. The effect of each protein over heme iron absorption was assessed. RESULTS: Intact heme uptake was higher than either heme plus albumin or digested heme plus albumin, but lower than digested heme. White meal exerted the highest inhibitory effect on hemin uptake. Heme iron uptake decreased in the presence of all legume extracts, but was not significantly different among them (one-way ANOVA, NS). Pure animal (collagen and casein) and vegetable (zein and glutelin) proteins increased heme iron uptake, except for gliadin. CONCLUSION: Animal and vegetable protein in general decreased heme iron uptake. However, purified animal and vegetable protein induce an increase in heme iron uptake.

Transepithelial heme-iron transport: effect of heme oxygenase overexpression.

BACKGROUND: Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. It is known that heme iron (heme-Fe) and inorganic iron are absorbed differently. Intracellularly, heme oxygenase-1 (HO1) participates in the cleavage of the heme ring producing biliverdin, CO and ferrous iron. Iron released from heme becomes part of labile iron pool, and it can be stored in ferritin or released through the basolateral membrane. The mechanism by which heme-Fe is metabolized within cells is not completely understood. OBJECTIVE: This study focused on the uptake and transport of heme iron and on the role of heme oxygenase-1 on heme iron metabolism. DESIGN: Caco-2 cells were incubated with different concentrations of heme-Fe. A full-length heme oxygenase-1 cDNA was expressed in Caco-2 cells and intracellular iron and heme-Fe content, heme uptake, heme and iron transport and heme oxygenase-1 immunolocalization were assessed in these cells. RESULTS: Heme-Fe was bioavailable and induced an intracellular increase in iron, ferritin and HO1 levels and a decrease in DMT1 expression. In cells overexpressing HO1, heme-Fe uptake and transepithelial Fe transport was higher than in controls. Most heme-Fe was metabolized to free iron, most of which was found mainly in the basolateral chamber. However, there is a fraction of heme that is delivered intact to the basolateral side. In a high heme-Fe condition, HO1 is found near the plasma membrane. CONCLUSIONS: These results suggest that heme oxygenase-1 catabolizes most of the heme-Fe and favors iron influx and efflux in intestinal cells.

[Some aspects of substantiation of an individual approach to the treatment of primary ischemic stroke].

The article presents assessment of the state of non-specific resistance of an organism by determination of SH-S-S coefficient relation (K(SU-S-S)) of blood and a spinal liquid in the first days of the disease and their change under influence of different medical products. Features of change of K(SU-S-S) level depending on severity of the clinical course of the disease. Peculiarities of reaction of SH-S-S indicator on pharmacological medications objectifies possibility of individual correction of SH-S-S level of patients with primary ischemic stroke.