Infantile pyknocytosis, a neonatal hemolytic anemia with Heinz bodies: A cohort study.

Infantile pyknocytosis (IP) is a rare, probably misestimated, cause of non-immune neonatal hemolytic anemia evolving in two phases: an initial phase with severe jaundice, followed by a second phase with hemolytic anemia, which may require neonatal intensive care. The diagnosis of IP is based on the transient presence on blood smear of hyperdense, contracted, and/or spiculated red blood cells (pyknocytes), associated with the spontaneous resolution of clinico-biological features and the exclusion of other causes. If the etiology remains undetermined, some contributing factors, such as oxidative stress, have been proposed. We report the description of 16 patients with IP aiming at clarifying the circumstances associated with the development of this acquired disorder. In the acute phase, the mean hemoglobin nadir and pyknocyte count were 7.8 g/dL and 11%, respectively, and strikingly, Heinz bodies were evident in 50% of the newborns, but in 100% after prolonged incubation (4 hours). A high proportion of Mediterranean or African ancestry was noted in newborns, as well as a significant number of peripartum events, such as respiratory distress. If the etiology of IP is certainly multifactorial, our series reinforces the role of oxidative stress, which may, at least in part, find origin in desaturation episodes in newborns.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BACKGROUND: In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology. OBJECTIVES: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS: Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing. RESULTS: Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu). CONCLUSIONS AND CLINICAL IMPORTANCE: This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH-binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

Hematological indices of injury to lightly oiled birds from the Deepwater Horizon oil spill.

Avian mortality events are common following large-scale oil spills. However, the sublethal effects of oil on birds exposed to light external oiling are not clearly understood. We found that American oystercatchers (area of potential impact n = 42, reference n = 21), black skimmers (area of potential impact n = 121, reference n = 88), brown pelicans (area of potential impact n = 91, reference n = 48), and great egrets (area of potential impact n = 57, reference n = 47) captured between 20 June 2010 and 23 February 2011 following the Deepwater Horizon oil spill experienced oxidative injury to erythrocytes, had decreased volume of circulating erythrocytes, and showed evidence of a regenerative hematological response in the form of increased reticulocytes compared with reference populations. Erythrocytic inclusions consistent with Heinz bodies were present almost exclusively in birds from sites impacted with oil, a finding pathognomonic for oxidative injury to erythrocytes. Average packed cell volumes were 4 to 19% lower and average reticulocyte counts were 27 to 40% higher in birds with visible external oil than birds from reference sites. These findings provide evidence that small amounts of external oil exposure are associated with hemolytic anemia. Furthermore, we found that some birds captured from the area impacted by the spill but with no visible oiling also had erythrocytic inclusion bodies, increased reticulocytes, and reduced packed cell volumes when compared with birds from reference sites. Thus, birds suffered hematologic injury despite no visible oil at the time of capture. Together, these findings suggest that adverse effects of oil spills on birds may be more widespread than estimates based on avian mortality or severe visible oiling. Environ Toxicol Chem 2018;37:451-461. © 2017 SETAC.

Hemoglobinas inestables / Unstable hemoglobins / Hemoglobinas instáveis

Las variantes estructurales de hemoglobina son, en su mayoría, el resultado de sustituciones puntuales de aminoácidos en una de las cadenas de globina. En muchos casos, estas hemoglobinopatías son inocuas, mientras que en otros determinan alteraciones en la estabilidad y función de la hemoglobina, ocasionando manifestaciones clínicas de severidad variable. En las hemoglobinas inestables, las alteraciones disminuyen la solubilidad, y así facilitan la formación de complejos de hemoglobina precipitada y desnaturalizada (Cuerpos de Heinz) que ocasionan el daño de la membrana y finalmente la destrucción prematura de los eritrocitos. Representan una rara etiología de anemia hemolítica congénita. Hasta la actualidad se han descrito alrededor de 150 hemoglobinas inestables diferentes, la mayoría de las cuales ocasionan hemólisis crónica, exacerbada por infecciones o la ingesta de medicamentos. Su diagnóstico puede ser difícil, sobre todo en pacientes no esplenectomizados. Su identificación requiere inicialmente de la sospecha clínica, pero dado que las pruebas básicas de laboratorio pueden no ser concluyentes e inducir un diagnóstico erróneo, el estudio molecular será necesario para la confirmación.

The structural hemoglobin variants mostly result from single amino-acid substitutions in globin chains. In many cases these are innocuous, but in others they may alter the stability or functional properties of hemoglobin, leading to clinical manifestations of variable severity. They represent a rare cause of congenital hemolytic anemia. Unstable hemoglobins cause alterations that reduce solubility, with increased tendency to Heinz Body formation, damaging red blood cell membrane and finally the premature destruction of red blood cells. Up to 150 unstable hemoglobins have been described; most of them cause chronic hemolytic anemia, aggravated by infections and or drugs. Diagnosis can be difficult, especially in non-splenectomized patients. Initially, identification requires a high degree of clinical suspicion, but due to the fact that basic laboratory tests might be inconclusive, molecular analysis is necessary for final confirmation.

As variantes estruturais de hemoglobina são, em sua maioria, o resultado de substituições pontuais de aminoácidos numa das cadeias de globina. Em muitos casos, estas hemoglobinopatias são inócuas, ao passo que em outros determinam alterações na estabilidade e função da hemoglobina, produzindo manifestações clínicas de severidade variável. Nas hemoglobinas instáveis, as alterações diminuem a solubilidade, facilitando a formação de complexos de hemoglobina precipitada e desnaturalizada (Corpos de Heinz) que ocasionam o dano da membrana e finalmente a destruição prematura dos eritrócitos. Representam uma rara etiologia de anemia hemolítica congênita. Até a atualidade foram descritas aproximadamente 150 hemoglobinas instáveis diferentes, a maioria das quais produzem hemólise crônica, exacerbada por infecções ou pela ingestão de medicamentos. Seu diagnóstico pode ser difícil, principalmente em pacientes não esplenectomizados. Sua identificação requer inicialmente da suspeita clínica, mas devido a que as provas básicas de laboratório podem não ser concluintes e induzir um diagnóstico errado, o estudo molecular será necessário para a confirmação.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies.

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil-injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Clinical oxidative stress during leprosy multidrug therapy: impact of dapsone oxidation.

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 µg/mL) and paucibacillary (0.662±0.123 µg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDT-supervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software.

Copper toxicosis in a Boer goat.

A 1-year-old female Boer goat was presented with a 1-day history of pigmenturia, anorexia, and shivering. Anemia was not present initially, but progressive hemolytic anemia developed subsequently and was characterized by the finding of Heinz bodies in both intact RBCs and in ghost cells and the presence of atypical fusiform RBCs. Plasma biochemical analysis revealed increased activities of aspartate aminotransferase and gamma-glutamyltransferase, hyperbilirubinemia, and azotemia. Histopathologic examination of a liver biopsy revealed necrosis of individual hepatocytes and intracytoplasmic rhodamine-positive granules, consistent with copper. Copper concentration in ante-mortem hepatic tissue was increased, and a diagnosis of copper toxicosis was made. Despite supportive therapy, the goat continued to decline and was euthanized. Necropsy findings included hepatic necrosis and hemoglobinuric nephrosis. Freshly collected specimens of liver and kidney had markedly increased copper concentrations. The mineral composition of the water, grass hay, and goat chow was evaluated, and toxins and significant mineral imbalances were not found. The underlying cause of the hepatic accumulation and subsequent release of copper remains unclear in this goat. Recently, Boer goats have been recognized as being prone to copper toxicosis and may be more susceptible than other breeds; similar to sheep, Boer goats may experience a hemolytic crisis secondary to copper toxicosis.

Peroxiredoxin I deficiency attenuates phagocytic capacity of macrophage in clearance of the red blood cells damaged by oxidative stress.

The role of peroxiredoxin (Prx) I as an erythrocyte antioxidant defense in red blood cells (RBCs) is controversial. Here we investigated the function of Prx I by using Prx I(-/-) and Prx I/II(-/-) mice. Prx I(-/-) mice exhibited a normal blood profile. However, Prx I/II(-/-) mice showed more significantly increased Heinz body formation as compared with Prx II(-/-) mice. The clearance rate of Heinz body-containing RBCs in Prx I(-/-) mice decreased significantly through the treatment of aniline hydrochloride (AH) compared with wild-type mice. Prx I deficiency decreased the phagocytic capacity of macrophage in clearing Heinz body-containing RBCs. Our data demonstrate that Prx I deficiency did not cause hemolytic anemia, but showed that further increased hemolytic anemia symptoms in Prx II(-/-) mice by attenuating phagocytic capacity of macrophage in oxidative stress damaged RBCs, suggesting a novel role of Prx I in phagocytosis of macrophage.

Allium species poisoning in dogs and cats: [review]

Dogs and cats are the animals that owners most frequently seek assistance for potential poisonings, and these species are frequently involved with toxicoses due to ingestion of poisonous food. Feeding human foodstuff to pets may prove itself dangerous for their health, similarly to what is observed in Allium species toxicosis. Allium species toxicosis is reported worldwide in several animal species, and the toxic principles present in them causes the transformation of hemoglobin into methemoglobin, consequently resulting in hemolytic anemia with Heinz body formation. The aim of this review is to analyze the clinicopathologic aspects and therapeutic approach of this serious toxicosis of dogs and cats in order to give knowledge to veterinarians about Allium species toxicosis, and subsequently allow them to correctly diagnose this disease when facing it; and to educate pet owners to not feed their animals with Allium-containg food in order to better control this particular life-threatening toxicosis.

Partial glutathione reductase deficiency as a cause of diverse clinical manifestations in a family with unstable hemoglobin (Hemoglobin Haná, ß63(E7) His-Asn).

Hemoglobin Haná [ß63(E7) His-Asn] is an unstable hemoglobin variant that was described in a Czech proband and her sister with Heinz body hemolytic anemia. The mother bearing the same mutation was asymptomatic; nevertheless, all three carriers had the same proportion of the mutant globin chains. Assessment of several erythrocyte antioxidant parameters revealed that both symptomatic children, unlike their asymptomatic mother, had significantly decreased glutathione reductase (GR) activity. Their GR activities were restorable in vitro by flavin adenine dinucleotide. The riboflavin supplementation improved their glutathione metabolism and ameliorated their hemolysis. Pre- and post-treatment assessment of the B(2) vitamers indicated suboptimal pre-treatment vitamin B(2) status in both children. This study provides evidence that partial GR deficiency may alter the clinical manifestation of an unstable hemoglobinopathy.

Effect of vitamin C, deferoxamine, quercetin and rutin against tert-butyl hydroperoxide oxidative damage in human erythrocytes.

The mature human erythrocyte, when submitted to oxidative stress, can demonstrate depletion of reduced glutathione, oxidation of the hemoglobin molecule and aggregation of complexes of iron close to the membrane. These can produce abnormalities in the erythrocyte membrane and hemolysis. The aim of this work was to study the antioxidative action of vitamin C (vit. C), deferroxamine (DFO) and the flavonoids quercetin and rutin in normal human erythrocytes, submitted to in vitro oxidative stress induced by tert-butylhydroperoxide ((t)BHP). Venous blood was collected in citrate-phosphate-dextrose (CPD) solution, as anticoagulant, from healthy adult individuals after informed consent. The erythrocytes were resuspended in PBS to obtain 35% globular volume, and then submitted to the oxidative action of (t)BHP for up to 30 min, with or without previous incubation for 60 min with vit. C, DFO, quercetin and rutin. Decrease in the GSH concentration, G6-PD and GR activities, and increase in the methemoglobin and Heinz bodies (HB) formation, occurred with the increase in (t)BHP concentration. (t)BHP did not effect on the membrane proteins detected by SDS-PAGE. Quercetin, partially prevented the GSH decrease and the formation of HB, but did not prevent MetHb formation from oxidative damage by (t)BHP. Rutin, after (t)BHP induction, prevented the GSH decrease and the formation of HB. Vit. C, had no influence on the depletion of GSH, inhibited partially the metHb formation, and it protected GR, but not G6-PD from oxidative damage by (t)BHP. DFO partially inhibited the metHb formation and GSH decrease, but it did not protect GR and G6-PD from oxidative damage by (t)BHP. The results obtained suggest that vit. C, DFO and the flavonoids quercetin and rutin contribute to the decrease in the oxidative stress caused by (t)BHP.

Oxidative process in erythrocytes of individuals with hemoglobin S.

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA

Clinical assessment of repeated propofol-associated anesthesia in cats.

OBJECTIVE: To assess the effects of repeated episodes of propofol-associated anesthesia on quality of recovery from anesthesia, clinical status, and erythrocyte physiology in cats. DESIGN: Original study. ANIMALS: 37 cats undergoing short-duration anesthesia for radiotherapy. PROCEDURES: Twice daily on 5 consecutive days, 13 cats with squamous cell carcinoma of the nasal planum (group 1) underwent anesthesia: first via administration of propofol or a midazolam (0.2 mg/kg [0.09 mg/lb])-propofol combination and then via administration of ketamine and midazolam each day (latter data were not analyzed). During a 19-day period, 24 cats with vaccine associated sarcoma (group 2) were anesthetized 12 times with propofol or a midazolam-propofol combination. Anesthesia was maintained with propofol in both groups. Hematologic analysis was performed before, during, and on completion of radiotherapy; changes in Hct and hemoglobin concentration between groups were compared. RESULTS: Mean duration of anesthesia was 8.1 minutes (range, 5 to 20 minutes); no adverse events were detected during recovery. Total dose of propofol administered did not differ between groups 1 (6.34 mg/kg [2.88 mg/lb]) and 2 (4.71 mg/kg [2.14 mg/lb]). Midazolam administration decreased the propofol dose by 26%. Overall decreases from baseline in Hct and hemoglobin concentration were not significantly different between the 2 groups, nor clinically important; however, compared with baseline, values in group 2 were significantly lower after 6 and 12 anesthetic episodes for both protocols. Heinz bodies were identified in low numbers in both groups during radiotherapy. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that repeated propofol-associated short-duration anesthesia does not lead to clinically relevant hematologic changes in cats undergoing short-duration radiotherapy.

Inhibition of Heinz body induction in an in vitro model and total antioxidant activity of medicinal Thai plants.

Herbs have been used for medicinal purposes for centuries and known to possess antioxidant properties that may help to reduce the risk of chronic diseases, cardiovascular disease, and cancer. We screen aqueous extracts from 20 medicinal plants in Thailand that were believed to possess anti-tumor activity, help immune-stimulating property and maintain blood stasis. The antioxidant activities were investigated in two bioassays. Firstly, we demonstrated inhibition of Heinz bodies induction caused by oxidants under in vitro condition. The percentages of Heinz body inhibition activity in plant extracts from Terminalia citrina, Cassia timoriensis, and Derris elliptica were the highest followed by Anamirta cocculus, and Oroxylum indicum respectively. In addition, we investigated total antioxidant activity in plant extracts by improved ABTS radical cation decolorization assay. The total antioxidant activity of the extract from Terminalia citrina was also the highest activity followed by Ficus pubigera, Derris elliptica, Anamirta cocculus, Caesalpinia sappan, and Oroxylum indicum respectively. Our results suggest medicinal Thai plants as valuable sources of antioxidants, which may have a potential anti-carcinogenic activity.

Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia.

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.

Aniline: early indicators of toxicity in male rats and their relevance to spleen carcinogenicity.

Early indicators of aniline hydrochloride (AH) toxicity were investigated in male Fisher 344 rats for 1 or 4 weeks at dietary dose levels of 10, 30 or 100 mg/kg body weight (bw)/day (actual intake at least 6, 17 and 57 mg/kg). The doses were based on earlier studies that had shown spleen toxicity and carcinogenicity in male rats at 100 mg/kg/day but not at 10 mg/kg/day. In the present study a dose-related formation of haemoglobin adducts and Heinz bodies was found from 10 and 30 mg/kg bw/ day, respectively, onwards. Signs of anaemia (decreased red blood cell counts and increased reticulocytes) were recorded from 30 mg/kg onwards. At 100 mg/kg, an overt haemolytic anaemia was associated with increases in serum transferrin concentration and total iron binding capacity in the blood reflecting major perturbations in iron metabolism. At this dose there was an increase in peripheral neutrophil leucocytosis in the blood, indicating an inflammatory process in the spleen. Histopathologic evaluation showed a focal perisplenitis and haemosiderin deposition in sinusoidal Kupffer cells of the liver at 100 mg/kg. These results corroborate the contention that carcinogenic doses of aniline cause early effects on haematological parameters, inflammatory reaction in the spleen and perturbations in iron metabolism as a result of haemolytic anaemia. Accordingly, the carcinogenicity of aniline may be linked to definable threshold-related processes.