Quantitative analysis of heme and hemoglobin for the detection of intravascular hemolysis.

BACKGROUND: Intravascular hemolysis is associated with massive release of hemoglobin and consequently labile heme into the blood, resulting in prothrombotic and proinflammatory events in patients. Though heme is well-known to participate in these adverse effects, it is not monitored. Instead, haptoglobin and hemoglobin serve as clinical biomarkers. The quantification of labile heme together with hemoglobin, however, should be considered in clinical diagnosis as well, to obtain a complete picture of the hemolytic state in patients. So far, quantification techniques for labile heme were not yet systematically analyzed and compared for their clinical application potential, especially in the presence of hemoglobin. RESULTS: Two commercial assays (Heme Assay Kit®, Hemin Assay Kit®) and five common approaches (pyridine hemochromogen assay, apo-horseradish peroxidase-based assay, UV/Vis spectroscopy, HPLC, mass spectrometry) were analyzed concerning their linearity, accuracy, and precision, as well as their ability to distinguish between hemoglobin-bound heme and labile heme. Further, techniques for the quantification of hemoglobin (Harboe method, SLS method, Hemastix®) were included to study their selectivity for hemoglobin and potential interference by the presence of labile heme. Both, indirect and direct approaches were suitable for the determination of a wide concentration of heme (∼0.02-45 µM) and hemoglobin (∼0.002-17 µM). A clear distinction between hemoglobin-bound heme and labile heme with one method was not possible. Thus, a novel combined approach is presented and applied to human and porcine plasma samples for the determination of hemoglobin and labile heme. SIGNIFICANCE: Our results demonstrate the need to develop improved techniques to differentiate labile and protein-bound heme for early detection of intravascular hemolysis. Here, we present a novel strategy by combining two spectroscopic methods, which is most reliable as an easy-to-use tool for the determination of hemoglobin and heme levels in plasma samples for the diagnosis of intravascular hemolysis and in basic biomedical research.

Detecting labile heme and ferroptosis through 'turn-on' fluorescence and lipid droplet localization post Fe2+ sensing.

Iron, a crucial biologically active ion essential for metabolic processes in living organisms, plays a vital role in biological functions, and imbalances in iron levels can lead to various diseases. In this study, we have developed two simple "turn-on" fluorescent probes, NOPy and NOCN, for the quick and selective detection of Fe2+ at nanomolar levels (LOD of 35 nM), accompanied by significant absorption and emission shifts, along with colorimetric demarcation. Both fluorophores exhibit an excellent "turn-on" emission response upon encountering Fe2+ in the cells. Flow cytometry and confocal fluorescence imaging studies demonstrate enhanced fluorescence signals in response to labile iron, efficiently detecting heme during erastin-induced ferroptosis. Interestingly, we also observed that the product formed after Fe2+ sensing localizes within the lipid droplets. These water-soluble and highly sensitive reactive probes, NOPy and NOCN, enable investigations of iron-dependent physiological and pathological conditions. The development of these probes represents an advancement in the field, offering a rapid and selective means for detecting Fe2+ with minimal cytotoxicity.

Zinc-protoporphyrin formation in nitrite-free Parma Ham and its relationship with intrinsic parameters and red color profile of processed hams.

A total of 134 fresh hams, assayed for Ferrochelatase (FeCH) activity and ultimate pH (pH48), were processed in compliance with the procedures established for PDO Parma ham and finally, analyzed for salt, moisture, Zinc Protoporphyrin IX (ZnPP), heme, iron and zinc contents, and proteolysis index (PI). The variation in ZnPP content was related to the intrinsic parameters of fresh and matured hams by a Partial Least Square Regression model. The most favorable factors on the formation of ZnPP were total iron content (representative of the initial hemoprotein content), and FeCH activity, demonstrating the main role played by these raw matter-specific predictors in the long matured dry-cured hams. To a lesser extent, zinc content and pH48 were involved with a positive and negative role, respectively. Salt content and PI of matured hams showed an inhibitory and a favorable influence, respectively, toward the ZnPP formation. Principal Component Analysis showed the associations between the sensory red color profile and the physicochemical traits of matured hams. The red color intensity increased in agreement with the red-violet and red-pink hues scores. The formation of a high amount of ZnPP was associated with the increased perception of the red-violet shade, with a lower lightness (L*) and Hue angle (h°). Moisture increase contributed to the shift in color perception to red-pink, while marked progress in PI strengthened the perception of the red-brown shade. ZnPP and final heme favored the red color of matured hams, although a high concentration of these pigments increased in particular the red-violet perception.

La Impossible Burger. La revolución de los alimentos en la UE / La Impossible Burger. La revolució dels aliments en la UE / The Impossible Burger. The food revolution in the EU

La difícil situación que atraviesa el planeta a causa, entre otros, del modelo agroalimentario actual, ha provocado que el sistema alimentario se convierta en uno de los grandes desafíos del siglo XXI y a la vez, en una nueva oportunidad económica. Empresas como Impossible Foods lideran una revolución tecnológica alimentaria. Su primer producto, la Impossible Burger, comercializado ya en los EEUU, es el objeto de esta tesina. Esta «Hamburguesa de plantas» pretende reemplazar a la hamburguesa de ternera convencional, proporcionando una experiencia sensorial idéntica o mejorada. Pero su ingrediente estrella, la heme, el cual marca la diferencia con el resto de hamburguesas vegetarianas, es la pieza clave que determinará el futuro comercial de este producto en Europa

La difícil situació del planeta a causa, entre altres factors, del model agroalimentari actual, ha provocat que el sistema alimentari es converteixi en un dels grans desafiaments del segle XXI i, alhora, en una nova oportunitat econòmica. Empreses com ara Impossible Foods lideren una revolució tecnològica alimentària. El seu primer producte, la Impossible Burger, comercialitzat ja als EUA, és l'objecte d'aquesta tesina. Aquesta «Hamburguesa de plantes» pretén substituir l'hamburguesa de vedella convencional, proporcionant una experiència sensorial idèntica o fins i tot millor. Però el seu ingredient estrella, la heme, que és el que marca la diferència amb la resta d'hamburgueses vegetarianes, és la peça clau que determinarà el futur comercial d'aquest producte a Europa

The difficult situation that the planet goes through due to the current agrifood model, among others, has caused the food system becomes one of the great challenges of the 21st century and nowadays, in a new economic opportunity. Companies like Impossible Foods lead a food technological revolution. Its first product, the Impossible Burger, already marketed in the United States, is the subject of this thesis. This «Burger of plants» aims to replace conventional beef burger, providing an identical or enhanced sensory experience. But its star ingredient, heme, which marks the difference with the rest of vegetarian burgers, is the key element which will determine the commercial prospects of this product in Europe

El grupo hemo y la ácido aminolevulínico sintasa 1 en la fisiopatología de los ataques agudos de porfiria / The haem group and aminolaevulinic acid synthase-1 in the pathophysiology of acute porphyria attacks

Las porfirias hepáticas agudas son 4 enfermedades raras causadas por deficiencias enzimáticas en la vía biosintética del grupo hemo. Se caracterizan por presentar síntomas neuroviscerales agudos potencialmente letales ante la presencia de factores inductores de la ALAS1. Estos factores pueden ser endógenos o exógenos tales como hormonas sexuales, ayuno, medicamentos, alcohol y tabaco, entre otros. La fisiopatología de los ataques involucra el incremento en la función de la ALAS1, la producción excesiva de precursores de porfirina y la alteración en la síntesis de hemoproteínas por la deficiencia relativa de hemo. En este artículo se revisa la interacción de esos mecanismos con algunos factores inductores, su papel en el origen de los síntomas neurológicos y cómo los tratamientos disponibles bloquean estos procesos (AU)

The acute hepatic porphyrias are a group of 4 rare diseases caused by enzymatic deficiencies in the haem biosynthetic pathway. They are characterized by presenting acute attacks of neurovisceral symptoms in presence of factors that increase the ALAS1 activity. Those factors could be endogenous or exogenous, such as sexual hormones, fasting, drugs, alcohol, tobacco, among other. The physiopathology of the attacks involves an increasing in ALAS1 function, excessive production of porphyrin precursors, and disturbances in hemoproteins synthesis due to the relative haem deficiency. The present paper is a review of the interaction of those mechanisms with some factors that induce ALAS1, their role in the origin of neurovisceral symptoms, and how the available treatments interfere with those processes (AU)

Ativação da heme oxigenase-1 e via da necroptose como mecanismos imunopatogênicos na infecção de macrófagos por leishmania infantum / heme oxygenase-1 activation and via the necroptose as immunopathogenics mechanisms for the infection of macrophages by Leishmania infantum

A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10...

Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α...

O papel do heme na infecção de neutrófilos humanos por Leishmania chagasi / The role of heme in human neutrophils infection by Leishmania chagasi

A Leishmaniose é uma doença que atinge milhões de pessoas em todo o mundo, sendo endêmica em muitas áreas. A doença pode apresentar diferentes manifestações clínicas, tais como a Leishmaniose Visceral (LV), que é a forma mais grave e letal, caso não seja tratada. As manifestações hematológicas são comumente associadas à LV, onde os mecanismos relacionados à hemólise e a presença do heme livre, podem interferir no comportamento de neutrófilos. Já foi descrito que o heme livre é uma molécula pró-inflamatória, com a capacidade de induzir migração e ativação dos neutrófilos. No entanto, o efeito do heme sobre neutrófilos humanos durante a infecção por Leishmania chagasi, ainda não foi explorado. A nossa hipótese é que o heme induz ativação de neutrófilos humanos favorecendo o processo inflamatório na infecção por L. chagasi. Nossos resultados mostraram que o heme induz a ativação e apoptose em neutrófilos humanos infectados com L. chagasi, seguido pela sobrevivência do parasita. Moléculas presentes na composição do heme, como a protoporfirina IX (PPIX) e o Fe+2, não alteram o status de ativação dos neutrófilos, mas mantêm o aumento do crescimento parasitário como observado em presença do heme. Além disso, o heme e o Fe+2 aumentam a produção do TGF-β e a atividade da SOD. A inibição farmacológica da enzima SOD com dietilditiocarbamato (DETC) reduz a taxa de proliferação da L. chagasi em neutrófilos infectados. Em conjunto, esses dados indicam que o heme e o Fe+2 podem contribuir como fonte nutricional e controlar o ambiente inflamatório, com a indução de TGF-β e SOD, permitindo a sobrevivência da L. chagasi em neutrófilos humanos. Esse estudo poderá abrir novas perspectivas para o entendimento dos mecanismos imunopatogênicos envolvendo neutrófilos e suas implicações no processo inflamatório da LV.

The Leishmaniasis is a disease that affects millions of people worldwide, being endemic in many areas. The disease may present different clinical manifestations, such as Visceral Leishmaniasis (VL), which is the most severe form and fatal if left untreated. The hematologic manifestations are commonly associated with VL, where the mechanisms related to hemolysis and the presence of free heme may modify the behavior of neutrophils. It has been reported that the free heme is a pro-inflammatory molecule with the ability to induce migration and activation of neutrophils. However, the effect of the heme on human neutrophils during infection by Leishmania chagasi yet hasn't been explored. Our hypothesis is that the heme induces activation of human neutrophils favoring the inflammatory process on the infection by L. chagasi. The results showed that the heme induces activation and apoptosis in human neutrophils infected with L. chagasi, followed by parasite survival. Molecules present in the composition of heme, as protoporfirin IX (PPIX) and Fe+2, do not alter the activation status of neutrophils, but maintain the increased parasite growth as viewed in the presence of heme. Furthermore, the heme and Fe+2 increase the production of TGF-β and SOD activity. The pharmacological inhibition of SOD with diethyldithiocarbamate (DETC) reduces the rate of L. chagasi proliferation in infected neutrophils. Together, these data indicate that the heme and Fe+2 may contribute as a nutritional source and control the inflammatory environment with the induction of TGF-β and SOD, allowing the survival of the L. chagasi in human neutrophils. This study may open new perspectives for the understanding of the immunopathogenic mechanisms involving neutrophils and its implications in the inflammatory process of the VL.

¿La hemozoína, presente y futuro? / ¿Hemozoin present and future

La malaria es una enfermedad causada por parásitos del género Plasmodium estos parásitos tienen un ciclo intraeritocítico en el hospedador vertevrado. En el glóbulo rojo, el parásito ingiere la hemoglobina, obteniendo aminácidos y formando hemozoína. La hemozoína es un un material microcristalino oscuro, de color marrón amarillento, insoluble en agua, no tóxico, producido en la vacuola parasitófora del Plasmodium; este compuesto producido por el Plasmodium carece de la toxicidad que tiene el grupo hemo para el parásito. Asimismo se ha evidenciado que la hemozoína es una sustancia inmuno moduladora que tiene diversos efectos, como mediar la activación y migración de neutrófilos, incrementar la producción de óxido nítrico, inducir la activación de mataloproteínas 9, inducir la secreción de diferentes mediadores proinflamatorios, alterar las funciones de los monocitos y macrófagos humanos, tales como el estallido oxidativo, eliminación de bacterias, presentación de antígenos y la habilidad de diferenciarse a células dendríticas funcionales; por lo que la hemozína tiene efectos duales, tanto activadores como supresores de la respuesta inmune. Asimismo, la hemozoína es unblanco terapéutico potente, ya que los fármacos que inhiban su formación provocan toxicidad al parasíto e incluso la muerte del mismo.

Malaria is a disease caused by parasites of the genus Plasmodium. These parasites have intraerythrocytic cycle in the vertbrate host. In the red cell, the parasite ingests hemoglobin, obtaining amino acids and formin hemozoin. The microcrystaline material hemozoin is a dark, yellowish brown, insoluble in water, nontoxic, produced in the Plasmodium parasitophorous vacuole, this compound produced by Plasmodiun lacks the toxicity that has heme to the parasite. It has also been shown that hemozoin is an immune modulating substance that has different ffects, mediating the neutrophils activation and migration, increased nitric oxide production, induce activation of metallproteinase-9, induce the secretion of various proinflammatory mediators, alter the funcions of human monocytes and macrophages such as oxidative burst, removing bacteria, antigen presentation and the ability to differentiate into functional dendritic cells, so the hemozoin has dual effects, both activators and suppressors of the immune response. Also, the hemozoin is a potent therapeutic target, since rugs that inhibit their formation causes toxicity to the parasite and even death itself.

Factores de riesgo y de protección para la anemia ferropénica en niños menores de 6 años: segundo premio poster: LIII Congreso Nacional de Pediatría 2007 / Risk and protection factor for iron deficiency anemia in children under 6 years of age: second reward poster: LIII National Congress of Pediatrics 2007

La anemia ferropénica es un problema de salud pública a nivel mundial. Los niños pequeños son más vulnerables a esta deficiencia. Determinar los factores de riesgo y protección para la anemia ferropénica en niños menores de 6 años. Estudio descriptivo, transversal. Se evaluaron 100 niños. Edad, género, estratificación social, tipo de lactancia, edad de ablactación, diagnóstico nutricional, características de la dieta (calorías, proteínas y hierro). Hemoglobina (Hb), Hematocrito (HTO), Volumen Corpuscular Medio (VCM), Hemoglobulina Corpuscular Media (HCM), hierro sérico. A las variables se les aplicó un análisis de regresión logística simple. 46 por ciento de los pacientes tenían anemia. Siendo la media para la edad de 19,2 meses, tiempo de lactancia materna exclusiva 5,2 meses, inicio de ablactación de 5,7 meses, hemoglobina de 9,9 g/dl. Se observa que en los niños de menor edad existe un mayor riesgo de presentar anemia. No se demostró una diferencia estadísticamente significativa entre los pacientes anémicos y no anémicos en relación al Gaffar Méndez Castellano y diagnóstico nutricional. Los factores de riesgo para la anemia con valores de Odds Ratio (OR) >1 fueron la edad menor de 24 meses, ausencia de lactancia materna exclusiva en menores de 6 meses, ablactación antes de los 5 meses, dietas hipocalóricas y el hierro sérico < 41 ug/dL. La dieta normoproteica resultó ser factor de protección (OR<1). Se evidenció la importancia de la lactancia materna y de una adecuada alimentación complementaria a partir del 5° mes de la vida como factores de protección para la anemia ferropénica en niños menores de 6 años.

Iron deficiency anemia is considered as a worlwide public health problem. Small children are more vulnerable to this deficiency. To determine the risk and protection factors for iron deficiency anemia in children under 6 years of age. This is a descriptive and cross-sectional study. 100 children were evaluated. Age, gender, social stratification, nursing type, complementary feedings, nutritional diagnosis, characteristic of the diet (calories, proteins and iron). Hemoglobin (Hb), hematocrit (HTO), mean corpuscular volume (VCM), mean corpuscular hemoglobin (HCM), total and fractional proteins, serum iron. 46% of patients wer anemic. Average values were: age 19,2 months, duration of exclusive breastfeeding 5.2 months, beginning of complementary feeding 5,7 months, hemoglobin 9.9 g/dl. Younger children had an increased risk of developin ganemia. There was not a statistically significant difference between anemic and non anemic patients in regard to Graffar and nutritional diagnosis. Risk factors for anemia (Odds Ratio-OR-values >1) were age under 24 months, lack of exclusive breastfeeding in children under 6 months, complementary feedings before 5 months of age, hypocaloric diet and serum iron <41 ug/dL. Normoproteic diets proved to be a protection factor (OR<1). Breastfeeding and appropriate complementary feedings after 5 months of age are protection factors for iron deficiency anemia in children under 6 years of age.

Cinética de ruptura do ferro heme em carne bovina (coxão mole - semi membranosus) submetida a diferentes tratamentos térmicos / Heme iron rupture kinetics in bovine meat (round beef - semi membranous muscle) submitted to different heat treatments

A concentração de ferro heme é um parâmetro de qualidade da carne, daí a importância de ser avaliado em carnes processadas. O objetivo deste trabalho foi avaliar a preservação do heme na cocção e no congelamento. Foram realizados dois experimentos com cortes de coxão mole (m. semi membranosus) moído, de origem bovina e em formato de hambúrgueres. Experimento1: a carne foi preparada em forno combinado a 150, 240 e 300’graus’C por 3, 5 e 10 minutos com umidade no forno de 10, 60 e 100%; em forno elétrico, a 150, 200 e 280’graus’C, por 6, 12 e 24 minutos e em chapa elétrica (200’graus’C) por 45, 120 e 240 segundos. Experimento 2: a carne foi congelada de forma lenta e rápida e armazenada por três meses. Foram determinadas as concentrações de ferro heme (FeH) pelo método de Hornsey, (1956) e modificado por Carpenter e Clark (1995), e avaliados parâmetros cinéticos da ruptura do heme. Esse efeito na carne cozida em todos os equipamentos mostrou ser bastante dependente da concentração. A redução da concentração de ferro heme durante a cocção seguiu cinética de segunda ordem. A partir da concentração de ferro heme inicial e das condições de cocção, as equações obtidas permitiram calcular as concentrações finais de ferro heme após preparação do coxão mole em forno combinado, chapa elétrica e forno elétrico com erros de 3,9; 5,7 e 17,9%, respectivamente. O congelamento, tanto rápido, como lento, não alterou a concentração do FeH. Com este estudo foi possível identificar as condições de preparo mais indicadas para a preservação do FeH em coxão mole, e os resultados poderão conferir um refinamento aos dados de tabelas de composição de alimentos, bem como servir como ferramenta de trabalho para profissionais que estão à frente de Unidades de Alimentação e Nutrição

Porfirias cutáneas / Cutaneous porphyrias

Las porfirias son un grupo heterogéneo de trastornos causados por un déficit parcial genético o adquirido de las enzimas que regulan la síntesis del grupo hemo. De acuerdo con la presencia o ausencia de fotosensibilidad cutánea, estas enfermedades pueden clasificarse en porfirias cutáneas y no cutáneas. Existen 5 tipos principales de porfirias cutáneas: la porfiria cutánea tarda (PCT); la porfiria variegata (PV); la coproporfiria hereditaria (CPH); la protoporfiria eritropoyética (PPE); y la porfiria eritropoyética congénita (PEC). La PV, CPH, PPE y la PCT tipo II son trastornos autonómicos dominantes con baja penetrancia. Las formas autosómicas recesivas (CEP y porfiria hepatoeritrocitaria, PHE) son trastornos de inicio precoz y muy raros. Las lesiones cutáneas en la PCT, la porfiria más frecuente, PV, CPH y PEC son similares: fragilidad mecánica, ampollas subepidérmicas, hipertricosis y pigmentación. PPE se caracteriza por una fotosensibilidad aguda y sin estas lesiones. Los ataques agudos de porfiria pueden ocurrir en la PV y CPH pero no en las otras porfirias cutáneas. La afectación hepática es una complicación infrecuente pero potencialmente fatal de la PPE. La PCT se asocia frecuentemente con enfermedad hepática crónica que a menudo es causada por el alcohol y suele ser leve. El diagnóstico clínico debe siempre confirmarse mediante análisis bioquimicos de porfirinas en orina, heces y sangre. La PCT puede ser tratada mediante sangrías repetidas para depleccionar los depósitos de hierro o mediante cloroquina a bajas dosis. El tratamiento de los otros tipos de porfiria cutánea es principalmente sintomático

The porphyrias are a heterogeneous group of disorders caused by genetically determined or acquired partial deficiencies of enzymes regulating heme biosynthesis. According to the presence or absence of cutaneous photosensitivity, these diseases can be classified into cutaneous and noncutaneous porphyrias. There are five main types of cutaneous porphyrias : porphyria cutanea tarda (PCT); variegate porphyria (VP); hereditary coproporphyria (He); erythropoietic protoporphyria (EPP); and congenital erythropoietic porphyria (CEP) Vp, HC, EPp, and one form of PCT (type II) are autosomal dominant conditions with low elinical penetrance. The autosomal recessive prophyrias (CEP and Hepatoerythropoietic porphyria, HEP) are rare disorders with early onset. The skin lesions in PCT (the commonest cutaneous porphyria), VP, HC, and CEP are similar: mechanical fragility, subepidermal bullae, hypertrichosis, and pigmentation. EPP is characterized by acute photosensitivity without these lesions. Acute attacks of porphyria may occur in VP and HC but not in other cutaneous porphyrias. Liver disease is an uncommon, potentially fatal, complication of EPP PCT is commonly associated with chronic liver disease, is often caused by alcohol and usually mild. The clinical diagnosis should always be confirmed by biochemical porphyrin analyses in urine, stool and blood. PCT can be treated by repeated venesection to deplete iron stores or with low-dose chloroquine. Treatment of the other cutaneous porphyrias is largely symptomatic