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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 AAASSS, by the increase in the content of Heinz bodies, methemoglobin, hemolysis, GSH depletion and lowering activities of the enzymes G6-PD and GR. The protective actions of the vitamins C and E for the oxidative stress induced by TBHP were observed for the erythrocytes in the lowering Heinz bodies, methemoglobin, hemolysis, and partial recovery of GSH more efficiently in AS and SS erythrocytes. Recovery was not observed in the levels of the activities of the enzymes G6-PD and GR, under the vitamins actions. The results obtained confirmed the higher susceptibility of the sickle erythrocyte to oxidation which necessitates precaution in the transfusion adequacy of AS erythrocytes. On the other hand, the protective effect of the vitamins C and E over the oxidative stress observed in erythrocytes AS and SS open perspectives for their use for treatment of patients with sickle cell anemias, as well as in the preservation of transfusional erythrocyte bag units.

The mechanism of hemolysis induced by onion poisoning in dogs was studied. Six adult, clinically normal Pekingese dogs were fed cooked onions at 30 g/kg body weight/day for 2 days. Blood samples were collected on days 1, 3, 5, 8, 12, 18 and 24 after onion administration, and urine was collected the day after bleeding. Red blood cell counts, hemoglobin and hematocrit were decreased from day 1, and significantly so on day 5 (P < 0.01), contrary to the results of white blood cell counts. So the plasma bilirubin levels and urobilinogen were increased on day 3 (P < 0.01) and day 4 (P < 0.01), respectively. The Heinz body counts were increased dramatically from day 1 (P < 0.01), peaking on day 3 (P < 0.01). Reticulocyte counts were increased from day 1 and the highest value was on day 8 (P < 0.01). Besides anemia, the following erythrocyte parameters were altered: erythrocyte glucose-6-phosphate dehydrogenase was reduced from day 1 and reached the lowest value on day 5 (P < 0.01); the reduced form of nicotinamide adenine dinucleotide phosphate was decreased on day 1 (P < 0.01); reduced glutathione was decreased from day 1 (P < 0.01) and the lowest value was on day 3 (P < 0.01); glutathione-peroxidase was increased on day 1, but decreased significantly on day 3 (P < 0.01); hydrogen peroxide was increased on day 1 (P < 0.01), then went down on days 3-12 (the undermost value on day 5, P < 0.01); catalase was risen dramatically on day 5 (the peak value, P < 0.01); malondialdehyde (MDA) was increased on days 1-8, (P < 0.01), and reached the peak value on day 5 (P < 0.01). Deformity of the erythrocyte membrane was decreased on days 1-12 and fluorescence polarization (rho) and microviscosity (eta) of the erythrocyte membrane were increased on days 1-12 (P < 0.01). There were positive correlations between MDA and rho as well as eta, with correlation coefficients of 0.908 and 0.922, respectively (P < 0.01), but there was a negative correlation between MDA and deformity index, with a correlation coefficient of -0.887 (P < 0.05). This study confirmed that onion poisoning in dogs causes hemolytic anemia.

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 vaccineassociated 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.

Polycyclic aromatic hydrocarbons (PAHs) in crude oil cause a range of adverse effects in oiled seabirds, one of the most common being hemolytic anemia via oxidative attack of erythrocytes by PAH metabolites resulting in hemoglobin leakage and formation of Heinz bodies. In such cases, haptoglobin and ferritin are up-regulated to sequester free Hb and iron in the circulation. We investigated these plasma proteins as biomarkers of PAH-induced Heinz body hemolytic anemia in oiled seabirds. Concentration ranges of PAHs, HAP and FT in plasma samples were 10–184 ng/ml, 0–2.6 mg/ml and 0–7.6 ng/ml, respectively. Dose–response relationships between plasma PAH exposure and haptoglobin and ferritin (FT) were investigated, and evidence of erythrocyte Heinz body formation studied in 50 oiled common guillemots stranded on the Norfolk Wash coast (East England). Haptoglobin was negatively correlated, and FT was positively correlated with PAH exposure. Heinz bodies were also observed confirming the toxic mechanism causing hemolytic anemia and counts were positively correlated with exposure. Our results support the application of these complementary biomarkers to assess hemolytic effects of oiling in wildlife biomonitoring, which also discriminate the influence of hemolytic versus inflammatory effects in oiled guillemots.

The about 400 million individuals worldwide suffering from a hereditary deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) may experience different degrees of haemolytic anaemia. Haemoglobin is present in very high concentrations in the erythrocyte cytoplasm, at risk of falling out of solution if the internal environment or the haemoglobin itself is changed. G6PD is a crucial enzyme producing reduced glutathione in the erythrocyte cytoplasm for the purpose of protecting haemoglobin against oxidative damage. The presence of unopposed oxidizing agents leading to oxidation of the sulfhydryl bridges between parts of the haemoglobin molecule decrease the solubility of haemoglobin, leading to precipitations called Heinz bodies. The laboratory investigation of G6PD deficiency is commonly done by a quantitative spectrophotometric analysis or by a rapid fluorescent spot test detecting the generation of NADPH from NADP. Genetic tests based on polymerase chain reaction detect specific mutations and may be used for population screening, family studies, or prenatal diagnosis.

Although aliphatic nitro compounds, aliphatic nitrates, and aliphatic nitrites have several features in common (nitrogen-oxygen grouping, explosiveness, methemoglobin formation), there are significant differences in their toxic effects. Some of their attributes are summarized. The esters of nitric and nitrous acid, whose nitrogen is linked to carbon through oxygen, are very similar in their pharmacological effects. Both produce methemoglobinemia and vascular dilatation with hypotension and headache. These effects are transient. None of the series has appreciable irritant properties. Pathological changes occur in animals only after high levels of exposure and are generally nonspecific and reversible. The nitric acid esters of the monofunctional and lower polyfunctional alcohols are absorbed through the skin. Information is not available on the skin absorption of alkyl nitrites. Members of both groups are well absorbed from the mucous membranes and lungs. Heinz body formation has been observed with the nitrates but not with the nitrites. Nitro compounds, like nitrates and nitrites, cause methemoglobinemia in animals. Heinz body formation parallels this activity within the series. Although some members are metabolized to nitrate and nitrite, there is no significant effect on blood pressure or respiration. As with the lower nitrates and nitrites, anesthetic symptoms are observed in animals during acute exposures, but these occur late. The prominent effect is irritation of the skin, mucous membranes, and respiratory tract. This is most marked with chlorinated nitroparaffins and nitroolefins. In addition to respiratory tract injury, cellular damage may be observed in the liver and kidneys. Skin absorption is negligible except for the nitroolefins. The nitramines have entirely different activity. RDX is a convulsant for humans and animals. Skin absorption, irritation, vasodilatation, methemoglobin formation, and permanent pathological damage are either insignificant or absent after repeated doses. Transient illness has been associated with the industrial use or manufacture of these materials, but fatalities and chronic intoxication have been uncommon. Some members of each group present extremely high fire and explosion hazards. Keywords: Aliphatic nitro compounds; Aliphatic nitrates; Aliphatic nitrites; Nitroolefins; Alkyl nitrites

Poisoning of Cattle Feeding on Allium ampeloprasum (Egyptian kurrat) Ten days after ingesting a large quantity of Allium ampeloprasum (kurrat), 10 one-year-old calves and 2 eight-year-old cows in a group of cattle were referred for voiding dark-red urine, generalized jaundice, anemic or icteric mucous membranes, lack of appetite, and lethargy. Hematologically, Heinz bodies – hemolytic anemia, polychromasia, anisocytosis and leukocytosis were detected. Biochemically, higher serum transaminases activities and bilirubin, urea and uric acid levels were detected. As well as, an induced lipid peroxidation and a perturbed antioxidant system; decreased glutathione level, and glutathione reductase and catalase activities were significantly detected. The treatment regimen of the poisoned animals included the parenteral administration of fluids, phosphorous and vitamins, and recovery occurred within one week after initiating the treatment. This is the first report describing Allium ampeloprasum toxicity in cattle with special reference to oxidative status.

The purpose of this study was to examine the changes of body composition and heinz body blood component after 3 days rapid weight loss(5.16% of total body weight) through combined method with total food restriction and dehydration. The result were as follows: The average weight showed a significant difference between before and after rapid weight loss(p after weight decrement in an average of before rapid weight loss, but the difference that noted did not show. The body fluid showed a significant difference between before and after rapid weight loss(p

Some clinical trials of vitamins C and E have neglected important design features. Our objective was to demonstrate a detailed design that includes essential elements for an effective study of these vitamins in vivo. While taking 400 IU (international units) of vitamin E, subjects took different dosages of vitamin C during three distinct periods. Dosages were 200 mg in food, 500 mg as supplements twice a day (500 × 2), and 1,000 mg as supplements twice a day (1000 × 2). Ten participants spent 3 weeks at each dosage before plasma was drawn on two consecutive days. Final samples were taken after a week with no supplementation. Selected by investigators at four institutions, endpoints were protein carbonyls, TBARs (thiobarbituric reactive substances), and Heinz body formation in RBCs (red blood cells). TBARs and protein carbonyls did not change significantly with dosage. However, Heinz body formation increased at either higher or lower intakes of vitamin C. Even with daily vitamin E, Heinz bodies were significantly fewer at 500 × 2. Results indicate that even with 400 IU vitamin E daily, it is possible to distinguish the effect of different levels of vitamin C with Heinz bodies. This effect may be due to pro-oxidant action of vitamin C or to prolonged survival of RBCs.

This article addresses results from a 4-wk inhalation exposure study in Wistar rats with the vapor and/or aerosol atmospheres of 2-chloro-4-toluidine. Groups of 10 rats/sex were nose-only exposed to mean analytical concentrations of 19.1, 115.1, and 702.3 mg/m3 using an exposure regimen of 6 h/day and 20-22 exposures within a time period of 4 wk. These concentrations were selected based on results from a repeated 5 x 6 h/day pilot study using concentrations of 27.1, 104.8, 381.6, and 1283.7 mg/m3. In a single 4-h exposure study at the maximum tested concentration of 7620 mg/m3, 1 of 10 female rats succumbed (no mortality in males), while no mortality occurred at 3293 mg/m3. In the 1- and 4-wk studies mortality occurred at 1283.7 and 702.3 mg/m3, respectively. Rats exposed for 4 wk to 702.3 mg/m3 displayed characteristic signs of toxicity that included cyanosis, respiratory distress, and significantly decreased body weights. Rectal temperatures were significantly decreased at 115.1 mg/m3 and above. Dark and enlarged spleens occurred at 702.3 mg/m3. At this concentration, prominent treatment-related effects included methemoglobinemia, reticulocytosis, red blood cells with Heinz bodies, decreased hemoglobin, hematocrit, and red blood cell counts. Borderline evidence of erythrocytotoxicty was noticed at 115.1 mg/m3 (based on a minimal increase in Heinz bodies). Spleen and liver weights were significantly increased at 702.3 mg/m3, whereas the thymus weight was decreased at 115.1 mg/m3 and above. Microscopic changes were found in the spleen (hemosiderosis) at 702.3 mg/m3. An atrophy of the olfactory epithelium in the nasal cavities occurred at 115.1 mg/m3 and above. Clinical pathology revealed changes pathognostic of hepatic effects, although microscopic examinations did not reveal any specific changes. The no-observed-adverse-effect level (NOAEL) of the 4-wk study was 19.1 mg/m3 and is based on the predominant atrophic changes of the olfactory epithelium and the minimal to borderline erythrocytotoxic effects at 115.1 mg/m3.

Objective—To characterize hematologic and clinical consequences of chronic dietary consumption of freeze-dried garlic at maximum voluntary intake in horses.Animals—4 healthy sex- and age-matched horses.Procedure—An initial garlic dose (0.05 g/kg, twice daily) was fed to 2 horses in a molasses carrier as part of their normal ration and was gradually increased to maximum voluntary intake (0.25 g/kg, twice daily) over 41 days. Dietary supplementation then continued for a total of 71 days. Two control horses were fed molasses with no garlic with their ration. Blood samples were collected weekly and analyzed for hematologic and biochemical changes, including the presence of Heinz bodies. Recovery of affected blood values was followed for 5 weeks after termination of dietary supplementation with garlic.Results—At a daily dose of > 0.2 g/kg, horses fed garlic developed hematologic and biochemical indications of Heinz body anemia, as characterized by increases in Heinz body score (HBS), mean corpuscular volume (MCV), mean corpuscular hemoglobin, platelet count, and serum unconjugated and total bilirubin concentrations and decreases in RBC count, blood hemoglobin concentration, mean corpuscular hemoglobin concentration, and serum haptoglobin concentration. Recovery from anemia was largely complete within 5 weeks after termination of dietary supplementation with garlic. Heinz body score and MCV remained high at the end of the 5-week recovery period.Conclusions and Clinical Relevance—Horses will voluntarily consume sufficient quantities of garlic to cause Heinz body anemia. The potential for garlic toxicosis exists when horses are chronically fed garlic. Further study is required to determine the safe dietary dose of garlic in horses. (Am J Vet Res2005;66:457–465)

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/12/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.

Two adult female alpacas were evaluated for acute onset of weakness, pale mucous membranes, and signs of depression of unknown etiology. Both alpacas had intravascular hemolysis, anemia, and Heinz body formation and had been fed wilted red maple leaves. Clinical signs developed several days after ingestion of the leaves. No other toxin exposure was reported, and no parasites were detected. Dietary copper and nitrate-nitrite concentrations were evaluated and determined to be within reference limits. Both alpacas continued to become profoundly anemic with continued hemolysis. This necessitated blood transfusion as a life-saving intervention; however, prior to whole blood being available, transfusion with bovine hemoglobin glutamer was necessary. Both alpacas recovered without complications.

Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low-molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca2+. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.

Thalassemia is relatively rare in Japan. Of 387 β-thalassemia cases of Japanese, homozygotes for β + - and β 0 -thalassemias were twenty-two (5.7%) and one (0.26%), respectively. The rest (94%) were of β - thalassemia trait. Ten different kinds of β-thalassemia mutations comprised about 80% of all cases. Half of these "common" mutations were unique to Japanese and another half were possibly from abroad. Sixty-nine α -thalassemia cases were analyzed, in which HbH and α - thalassemia trait comprised nineteen and fifty, respectively. Most of the α o - and α + -thalassemia chromosomes were of Southeast Asian type (--SEA) and -α 3.7 type, respectively. The frequency of α + - thalassemia as well as triplication of α -globin gene was high in northern Japan. Recent increase in the number of immigrants from Southeast Asia seems to raise the number of α -thalassemia found in Japan. They comprise at least 20% of the α -thalassemia. Six mutants classified into dominant-type β-thalassemia were found. All of them exhibited moderate anemia and marked anisopoikiocytosis. Heinz body varied in degree from copious to rare or even absent. Any mutation at initiation codon demonstrated marked microcytosis and erythremia. The breakpoint determination for large deletion-type thalassemia became feasible by estimation of gene dosage and PCR. Thus, the precise breakpoints for Filipino-type α o -thalassemia (--FIL) and Japanese type-2 δβ-thalassemia were disclosed. The genetic diagnosis for these thalassemias are now readily conducted by gap PCR. The characterization of several new large deletions is being carried out.

Anemia represents a common problem encountered in daily clinical practice, affecting 3.4 million Americans. Hence, a structured approach to evaluating anemia is highly relevant in primary care. Multichannel automated analyzers have revolutionized the work of performing complete blood counts (CBCs). However, when interpreting results, clinicians must be aware of limitations encountered with these technologies, which can yield spurious red blood cell (RBC) values in certain clinical circumstances. Automated analyzers identify subpopulations of RBCs that are unexpectedly small or large or have unexpectedly low or high hemoglobin concentrations, thereby signifying aberrant RBC morphologies for review on peripheral blood smear. Results of various blood chemistries help to refine or confirm diagnostic considerations suggested by the CBC, reticulocyte count, and peripheral blood smear. Although tempo of anemia development may strongly support bleeding or hemolysis as the cause of anemia, kinetic changes in RBC mass, even due to these mechanisms, are often more subtle, suggesting possible underproduction causes of anemia. Abnormalities in white blood cell (WBC) counts, platelet counts, and WBC differentials may suggest disorders of trilineage hematopoiesis, although multiple competing factors may coexist, with certain factors affecting RBCs independent of those affecting WBCs and/or platelets. To focus diagnostic considerations, clinicians should consider anemia etiologies categorized by RBC size (mean cell volume, MCV) and morphology (eg, spherocytes, bite cells, schistocytes, target cells, teardrops). These categories include the microcytic, normocytic, and macrocytic anemias. Each of these categories are briefly reviewed, and case presentations are provided to illustrate specific points.

S-adenosylmethionine (SAMe) is reported to have hepatoprotective and antioxidant functions. Acetaminophen (paracetamol) was used to induce oxidative damage in cats, and to then determine the effect of SAMe treatment on erythrocyte morphology, PCV, liver histopathology, thiobarbituate reacting substances (TBARS), reduced glutathione (GSH), and oxidised glutathione (GSSG).Cats receiving acetaminophen had a significant increase in methemoglobin and Heinz body production. A significant effect for the interaction of time and treatment was found for Heinz body production and changes in PCV. No significant changes were found in blood or hepatic TBARS. Blood GSH increased significantly in all cats, while the blood GSH:GSSG ratio tended to increase the most in cats given acetaminophen only. The hepatic GSH:GSSG ratio tended to increase in cats given SAMe and decrease in cats given acetaminophen, but this effect was not significant.SAMe protected erythrocytes from oxidative damage by limiting Heinz body formation and erythrocyte destruction and maybe useful in treating acetaminophen toxicity.

Saturation transfer phenomena from irradiated protein protons to observed water protons in packed human red blood cells (RBCs) with normal or unstable hemoglobin (Hb), i.e. Hb Yokohama and Hb Koeln, were studied using intermolecular cross-relaxation rates [CR; 1/T(IS)(H(2)O)], action spectra [[1-(I(infinity)/I(0))] vs f(2) (ppm), where I(0) and I(infinity) are the longitudinal magnetization of observed water protons before and after long-time f(2)-irradiation, respectively], CR spectra [CR vs f(2) (ppm)] and CR ratio vs f(2) (ppm) with f(2)-irradiation from -100 to 100 ppm at gammaH(2)/2pi of 69 or 250 Hz. RBCs (Hb Yokohama) exhibited many large Heinz bodies and strongly impaired filterability, while RBCs (Hb Koeln) showed few microscopically typical Heinz bodies and virtually normal filterability. However, increases in CR values for RBCs (Hb Koeln) and RBCs (Hb Yokohama), monitored by f(2)-irradiation below approximately -6 and above approximately 14 ppm, clearly indicated marked increases in association or aggregation of unstable Hb in RBCs compared with those in normal RBCs. CR values, monitored between approximately 0 and approximately 10 ppm, were related to not only association or aggregation of unstable Hb but also amounts of water in RBCs. Aggregation or association of unstable Hb exhibited greater effects on CR values compared with those of methemoglobin formation.

Objective: To report the use of ultrapurified bovine hemoglobin (PBHg) in the treatment of red maple toxicosis in horses. Summary: The use of PBHg in a miniature horse and a pony with clinical signs of red maple toxicosis is described. Initial treatment of the horses included blood transfusion in Case 1, and intravenous crystalloid fluids, nasogastrically administered mineral oil, and activated charcoal in both cases. Both cases received PBHg (16 and 11 ml/kg, respectively) when the hematocrit dropped below 10%. Clinical stabilization of the horses (reflected by improved vital signs and mucous membrane color, increase in PvO2, and increase in plasma hemoglobin) was temporary, lasting 12 and 42 hours, respectively. A whole blood transfusion was given 16 hours after the infusion of PBHg in Case 1 and 42 hours after the infusion of PBHg in Case 2. The horses were discharged on days 15 and 17, respectively. Both were stable on discharge and were reported to be doing well 1 month after discharge. New information provided: Ultrapurified bovine hemoglobin is shown to be safe in the treatment of red maple toxicosis. Ultrapurified bovine hemoglobin infusion resulted in temporary improvement in oxygen (O2) delivery as assessed by clinical and laboratory parameters. The use of PBHg may provide an ‘oxygen bridge’ in an acute hemolytic crisis in the horse. It may allow additional time in the treatment of red maple toxicity for the acute hemolysis to subside and for metabolism of the toxin to occur, thus providing a more optimal environment for an effective whole blood transfusion.

Hb Presbyterian is a variant hemoglobin that carries Lys at Asn-108 of beta-globin. This variant Lys(beta108) residue enhances the stability of Hb in the deoxy-state, conferring the low affinity for oxygen-binding in vitro. In the present study, we generated mutant mice carrying the Presbyterian mutation (Asn(beta108)-->Lys) at the beta-globin locus by a targeted knock-in strategy. Heterozygous mice showed the expression of Hb Presbyterian in 27.7% of total peripheral blood without any hematological abnormalities, which well mimicked human cases. On the other hand, homozygous mice exclusively expressed Hb Presbyterian in 100% of peripheral blood associated with hemolytic anemia, Heinz body formation, and splenomegaly. Hb Presbyterian showed instability in an in vitro precipitation assay. Erythrocytes from homozygous mice showed a shortened life span when transfused into wild-type mice, confirming that the knocked-in mutation of Lys(beta108) caused hemolysis in homozygous mice. This is the first report on the hemolytic anemia of unstable hemoglobin in an animal model. These results confirm the notion that the higher ratio of an unstable variant beta-globin chain in erythrocytes triggers the pathological precipitation and induces hemolysis in abnormal hemoglobinopathies.

A intoxicação por cebola é relatada em várias espécies animais em muitas partes do mundo. O princípio tóxico (n-propil dissulfito) presente na cebola causa a transformação da hemoglobina em metemoglobina. Para estudar os achados laboratoriais, de necropsia e histopatológicos da intoxicação por cebola em gatos, cinco gatos de quatro meses de idade receberam cada um uma dose única de 10g/kg de cebola desidratada por via oral. Um outro gato de mesma idade não recebeu a refeição com cebola e serviu como controle. Todos os cinco gatos desenvolveram sinais clínicos da toxicose; um deles morreu dentro de 24 horas após a ingestão da cebola. Os sinais clínicos incluíram apatia, taquicardia, taquipnéia e cianose. Os achados laboratoriais se caracterizavam por anemia hemolítica associada a corpúsculos de Heinz e metemoglobinemia. Os principais achados de necropsia foram esplenomegalia e sangue de cor marrom. Os achados histopatológicos foram hemossiderose e hematopoese extramedular no baço e fígado.

S-Nitrosated hemoglobin is remarkably stable and can be cycled between deoxy, oxygenated, or oxidized forms without significant loss of NO. Here we show thatS-nitrosation of adult human hemoglobin (Hb A0) or sickle cell Hb (Hb S) results in an increased ease of anaerobic heme oxidation, while anions cause redox shifts in the opposite direction. The negatively charged groups of the cytoplasmic domain of Band 3 protein also produce an allosteric effect on S-nitrosated Hb. Formation and deoxygenation of a SNO-Hb/Band 3 protein assembly does not in itself cause NO release, even in the presence of glutathione; however, this assembly may play a role in the migration of NO from the red blood cells to other targets and may be linked to Heinz body formation. Studies of the anaerobic oxidation of Hb S revealed an altered redox potential relative to Hb A0 that favors met-Hb formation and may therefore underlie the increased rate of autoxidation of Hb S under aerobic conditions, the increased formation of Heinz bodies in sickle cells, and the decreased lifetime of red cells containing Hb S. A model for the interrelationships between the deoxy, oxy, and met forms of Hb A0 and Hb S, and their S-nitrosated counterparts, is presented.

Methylene blue trihydrate is used widely as a dye and therapeutic agent. Methylene blue was administered by gavage to 30 animals/sex/dose group in a 0.5% aqueous methylcellulose suspension at doses of 0, 25, 50, 100, and 200 mg/kg. Blood samples from 10 animals/sex/dose group were collected at the end of study weeks 1, 6, and 13. Methylene blue treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change was a dose-related increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice appeared to be more sensitive than rats to the formation of Heinz bodies and the development of anemia that was characterized by a decrease in hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated mice and in the 100 mg/kg (males only) and 200 mg/kg rats at necropsy. There was a dose-related increase in absolute and relative spleen weight for both species. Microscopic examination revealed increased splenic hematopoiesis in all mice treatment groups and in rats at the 50 mg/kg dose level and above. Splenic congestion and bone marrow hyperplasia were also observed in these rat-dose groups. Mice at the higher doses showed hematopoiesis in the liver and accumulation of hemosiderin in Kupffer cells. These gross and microscopic findings are consistent with the development of hemolytic anemia. A dose-related increase in the reticulocyte count during study weeks 6 and 13 suggested a compensatory response to anemia.

Objective: To report an unusual manifestation of acetaminophen intoxication in a dog. Case Summary: A Miniature Pinscher was presented for evaluation of lethargy and facial swelling after ingestion of 500–750 mg/kg of acetaminophen. Laboratory testing revealed eccentrocytes, Heinz bodies, hemoglobinemia, and a declining packed red cell volume consistent with oxidative damage to the erythrocytes. Hepatocellular damage, as monitored by serum alanine aminotransferase, was not documented. The dog responded well to therapeutic intervention, and was discharged from the hospital. The dog returned to the hospital 2 days later with clinical signs and diagnostic tests consistent with acute keratoconjunctivitis sicca, which was assumed to be due to a toxic etiology. The dog responded well to therapy, regaining normal levels of tear production within six weeks. New or Unique Information Provided: Acetaminophen intoxication may be associated with severe erythrocytic oxidative damage in the absence of detectable hepatic injury in dogs. Acetaminophen intoxication may have the potential to cause acute keratoconjunctivitis sicca in dogs. (J Vet Emerg Crit Care 2001; 11(2): 123–126)

Objective—To determine the effectiveness of 3 antioxidants in preventing Heinz body anemia in cats. Design—Prospective study. Animals—44 specific-pathogen-free healthy cats. Procedure—Cats were housed individually, divided randomly into 4 groups, and given the following orally every 12 hours: empty gelcaps (control cats), Nacetylcysteine (NAC, 100 mg/kg of body weight), vitamin E (d,l-α-tocopherol; 400 IU), or ascorbate (250 mg). After 2 weeks, Heinz bodies were induced by dietary onion powder (OP; 1% or 3% of dry matter) or propylene glycol (PG, 8% wt/vol in drinking water) for an additional 3 weeks. Intake of treated water or food was recorded daily. Body weight, PCV, Heinz body and reticulocyte percentages, reduced glutathione concentration, and total antioxidant status were measured twice weekly in all cats. Results—Heinz body percentage and degree of anemia did not differ significantly among cats receiving antioxidants and control cats except in cats that ingested water containing PG, in which antioxidant supplementation was associated with a decrease in water intake. Of cats that were fed a diet that contained OP, cats that received NAC had significantly higher reduced glutathione concentrations, compared with other cats in the experiment. Total antioxidant status did not consistently correlate with antioxidant supplementation or type of oxidant administered (ie, OP or PG). Conclusions and Clinical Relevance—Although the effect of antioxidant supplementation on Heinz body anemia in cats was minimal, antioxidants may have subclinical biochemical effects such as GSH sparing that may be important against milder forms of oxidative stress. (Am J Vet Res 2001;62:370–374)

Visceral leishmaniasis is accompanied by severe anemia and pancytopenia. Reactive oxygen species are known to contribute to the pathogenesis of several red blood cell (RBCs) disorders. The present study reveals the extent of oxidative stress and the efficacy of the primary antioxidant system in erythrocytes of hamsters in the progressive anemic response at different stages of leishmanial infection. Increased intracellular precipitation of Heinz bodies secondary to oxidative denaturation of hemoglobin and enhanced formation of malonyldialdehyde suggest oxidative damage of erythrocytes, both in the hemoglobin and cell membrane, respectively. Decreased activities of superoxide dismutase and catalase in the infected animals indicate the generation of O2 * – and H2O2, which in turn may produce the highly reactive *OH species. Decreases in the reduced glutathione level along with the decreased activities of glutathione reductase and glutathione peroxidase point to a deficient antioxidant defense system during the post-infection period. Accentuated degradation of both cytoskeletal and integral membrane proteins after 3 months of infection may eventually lead to membrane destabilization and early lysis of erythrocytes in experimental visceral leishmaniasis.

Objective—To determine the effect of a commercial bioflavonoid antioxidant on acetaminophen-induced oxidative injury to feline erythrocytes. Design—Randomized controlled study. Animals—45 healthy age-matched cats. Procedure—Cats were assigned to 3 experimental groups. Groups 1 and 3 received a bioflavonoid antioxidant (10 mg/d) orally for 2 weeks. Groups 2 and 3 received an oxidative challenge with acetaminophen (90 mg/kg [41 mg/lb] of body weight, PO) on day 7. Packed cell volume, percentage of erythrocytes with Heinz bodies, blood methemoglobin concentration, and blood reduced and oxidized glutathione concentrations were determined at various times during the 2-week study period. Results—Adverse effects were not associated with bioflavonoid antioxidant administration alone. Acetaminophen administration resulted in a significant increase in methemoglobin concentration in groups 2 and 3; differences were not detected between these groups. Heinz body concentrations in groups 2 and 3 increased after acetaminophen administration; however, the increase in cats that received the antioxidant was significantly less than in group-2 cats. Total blood glutathione concentrations did not change significantly in groups 2 and 3 after acetaminophen administration; however, ratio of reduced to oxidized glutathione concentration increased significantly after administration in group-2 cats, compared with group-3 cats. Conclusions and Clinical Relevance—Oral administration of bioflavonoid antioxidants to cats at risk for oxidative stress may have a beneficial effect on their ability to resist oxidative injury to erythrocytes. (J Am Vet Med Assoc 2000;217:1157–1161)

vated methemoglobin levels and the occurrence of Heinz bodies are frequently, but not always, perceptible indicators of oxidative damage, often having disappeared by the time hemolysis is obvious. Often, circulating red blood cells with apparent pieces missing from their perimeter appear on blood smears.5 Hemoglobinemia and hemoglobinuria are usually of moderate degree during the explosive episode, but typically hemolysis is limited in duration, because only relatively old cells are vulnerable. The more nearly normal G-6-PD activity found in younger red blood cells, which become more abundant in the circulation in response to hemolysis, protects them from excessive oxidative damage. Consequently, the red blood cell count may return to normal even though administration of the offending drug is continued, and diagnosis of a G-6-PD deficiency by assay of red blood cell enzyme activity may be difficult in this equilibrium phase when reticulocytosis is marked.6 Confirmation of the clinical diagnosis often must await the return of the red blood cell age distribution to normal, which is what was found in second test sample in this case. At least 100 million persons in the world have red cell G-6-PD deficiency,3 so that efforts to screen populations at high risk using 1 of many simple screening tests for this deficiency should be encouraged. Screening, performed when the patient is well, can serve to forewarn both patient and physician of increased susceptibility to such things as oxidant drugs, fava beans, and infections.

Polyploidy is one of Nature’s strategies to create diversity among fauna and flora, resulting in new species. We have used light, scanning and transmission electron microscopy to perform morphometric analyses in maturing erythroid cells of the cryptic species diploid and tetraploid Odontophrynus americanus frogs. Normal blood of both specimens contained 97%–99% erythrocytes and 1%–3% reticulocytes, besides thrombocytes and leucocytes. Mature erythrocytes were flattened, ellipsoidal, nucleated, with cytoplasm rich in haemoglobin. Five days after being made anaemic, 15% and 33% of diploid and tetraploid red blood cells respectively, were in an immature stage, basically proerythroblasts. These cells were also seen at the 10th day, in addition to basophilic and polychromatophilic erythroblasts. By day 15 a higher number, 75% and 89% of reticulocytes in earlier maturation stage was found, respectively. At day 20 of recovery from anaemia, there were 63% and 85% of reticulocytes, respectively, most in an advanced stage of maturation. The number of immature cells then gradually decreased at days 30 and 50. Cytoplasmic inclusions similar to Heinz bodies were found in these cells associated with RNA or RNP. Morphometric analysis showed that the tetraploid erythroid cells synthesise 30% more ribosomes than the diploid erythroid cells. The density of ribosomes/μm2 allowed these cells to be classified into seven classes: proerythroblasts, basophilic erythroblasts I and II, polychromatophilic erythroblasts I and II, reticulocytes and erythrocytes. Such morphometric strategy suggested that gene activity was more intense in the tetraploid maturing erythroid cells, despite the marked tendency of these tetraploid cells towards diploidisation of the genome expression.

Venous blood obtained from healthy donors and from patients suffering from breast cancer have been treated with acetylphenylhydrazine (APH) for different time. Mossbauer spectra of the packed red cells have been recorded and compared. The largest difference occurs after 50 min of treatment with APH where the patient samples show a broad spectral pattern indicating an advanced hemoglobin oxidation. These results may have some relevance in early cancer diagnosis.

Of the numerous beta-thalassemic mutations linked or unlinked to the beta-globin gene, all invariably cause a decrease in or an absence of structurally normal beta-globin mRNA when assayed. Here we report an anemic patient with an elevated alpha-/beta globin synthesis ratio of 2.0 in his reticulocytes. The patient's blood film showed marked red cell anisopoikilocytosis, microcytosis, and hypochromia, consistent with a typical beta-thalassemic trait phenotype. Acid-eluted erythrocytes contained numerous Heinz bodies. Molecular analysis of the patient's reticulocyte mRNA indicated that, compared to normal controls, there was a 3-fold elevation of beta-globin mRNA when assayed by RT-PCR and a 1.5-fold elevation of beta-globin mRNA when assayed by RNA slot blotting. The level of alpha-globin mRNA was normal when compared to that of normal adult controls. Extensive structural analysis of the beta-globin mRNA and gene by sequencing of RT-PCR and PCR products, respectively, did not detect any mutations. Tryptic mapping of purified beta-globin chains also did not show any abnormal tryptic fragments. These data indicated that a relative insufficiency of structurally normal beta-globin mRNA was not a cause of this beta-thalassemic phenotype. Therefore, the lesion that caused this particular thalassemic phenotype is not linked to the beta-globin allele.

Cystalysin, a 46-kDa protein isolated from the cytosol of Treponema denticola, was capable of both cysteine dependent hemoxidation and hemolysis of human and sheep red blood cells. The activities were characteristic of a cysteine desulfhydrase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting analysis of the interaction of cystalysin with the red blood cells revealed an interaction of the protein with the red blood cell membrane. Substrates for the enzyme (including L-cysteine and beta-chloroalanine) enhanced the interaction, which occurred with both whole red blood cells as well as with isolated and purified red blood cell ghosts. SDS-PAGE and western immunoblotting employing anti-hemoglobin serum revealed that, during the hemoxidative events, the hemoglobin molecule associated with the red blood cell membrane, forming putative Heinz bodies. Spectrophotometric analysis of the hemoxidative events (cystalysin + cysteine + red blood cells) revealed a chemical modification of the native hemoglobin to sulfhemoglobin and methemoglobin. Hemoxidation also resulted in the degradation of both the red blood cell alpha- and beta-spectrin. The results presented suggest that the interaction of cystalysin with the red blood cell membrane results in the chemical oxidation of the hemoglobin molecule as well as an alteration in the red blood cell membrane itself.

Free-ranging marine birds are severely impacted by petroleum released into the environment. Although petroleum can affect many organ systems, oxidative damage to red blood cells (RBC) and development of Heinz body anaemia is the only known mechanism of RBC damage in oil-exposed marine birds. Rhinoceros auklets (Cerrorhinca monocerata) were orally exposed to 0, 2.5 or 10 ml of Prudhoe Bay crude oil/kg body weight for five consecutive days by gavage tube. No statistically significant differences between treatment groups were evident for the following blood parameters: packed cell volume (PCV); haemoglobin concentration (Hb); mean corpuscular haemoglobin concentration (MCHC); reticulocyte percentage: fibrinogen concentration; white blood cell count (WBC); and cell counts of heterophils, lymphocytes, monocytes, eosinophils and basophils. After petroleum exposure, blood samples from auklets did not have any evidence of haemolysis, oxidative RBC damage or Heinz body formation as determined by new methylene blue staining of blood smears. Anaemia developed in both oil-exposed and unexposed auklets within 3 weeks of being captured and placed in captivity. Anaemia persisted throughout the duration of the study (77 days). Statistically significant differences over time were identified for PCV, Hb concentration, MCHC, reticulocyte percentage, fibrinogen concentration and lymphocyte, eosinophil and basophil numbers in auklets, without regard to petroleum exposure status. A mild transient regenerative response was noted after gavaging birds with either petroleum or sterile saline, however, the PCV never returned to levels measured immediately after capture. It is likely that anaemia associated with petroleum exposure in seabirds is multifactorial and may be associated with the sedentary nature of captivity, a variety of captivity associated stressors and possibly, the age of affected birds. Furthermore, neither inflammatory nor acute phase responses were consistent indicators of petroleum ingestion.

The mechanism of onion-induced hemolytic anemia in ruminants was investigated. The ether-extract obtained from the mixture of rumen fluid and onion juice incubated at 38.5 degrees C for 9 hr induced oxidative damage in sheep erythrocytes in vitro, indicating the production of certain oxidants in the mixture. The increase of the oxidative effect in the mixture was inhibited completely by the removal of rumen microorganisms and partly by treatment with antibiotics and by oxygen gas. The sheep fed onions (50 g/kg body weight/day) for 15 days developed more severe Heinz body hemolytic anemia than did the sheep fed the equivalent amount of onions with 5 g/day ampicillin sodium salt. The results indicated that certain rumen bacteria appear to be involved in the onset of onion-induced hemolytic anemia in sheep.

Presented is an African giant rat (Cricetomys gambianus) following zinc ingestion. The sick rat was lethargic, withdrawn, had soft, mucus-impregnated faeces and diahorrea, foot twitching and icterus. Comparative age, sex and body weight (b.wt.)-matched analyses were made with a healthy giant rat. Twelve-hourly Urine volume (UV), Haematocrit (Hct), urinary glucose, plasma zinc and Alkaline Phosphatase (ALP) were performed over an 8-week period. Full blood counts were performed and differential WBC counts and microscopic observations were made on blood smears obtained from both healthy and sick rats. Consecutive blood samples were drawn at the end of each week (Weeks 2-6 treatment; Weeks 7-8 post-treatment). Treatment involved oral vitamin B12 supplement at 4 µg / day and 2m diethylenetriaminepentaacetic acid (DTPA) intramuscular injections at 1m / 450g b.wt. / 5 wks (Week 2 - 6). Day 1 showed neutropaenia, Heinz bodies on RBCs (reticulocytes and immature forms). Zinc (Day 1 - end Week 7), glucose (Day 1 - end Week 4), ALP (Day 1 - Week 4) and UV were elevated (Day 1 - end Week 6). Indications of moderate zinc toxicosis following ingestion and stress-associated glucosuria were concluded.

The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective enzymes G6PDH and GR. Since these enzymes play a key role in the protection of erythrocytes against oxidative stress a risk of potentiation during mixed exposure does exist.

Thirteen-week oral repeated dose toxicity of ecabapide, a gastroprokinetic drug, was investigated in dogs at dosage levels of 50, 175 or 600 mg/kg, and in rats at dosage levels of 25, 100, 400 or 1600 mg/kg. In dogs, vomiting, aqueous salivation, body weight gain inhibition, and hemolytic anemia, together with an increase in Heinz body formation, were observed at 175 and/or 600 mg/kg. Histological examination revealed enhanced hemosiderin deposition in the liver and spleen, retention of erythrocytes in the splenic sinus and enhanced erythropoiesis in bone marrow at 175 and/or 600 mg/kg. In the rat study, although increases in serum total protein, albumin and calcium, as well as increased liver and kidney weights, were observed at 400 and/or 1600 mg/kg, no obvious morphological changes were seen. The hemolytic anemia and an increased Heinz body formation were not observed in rats, indicating a species difference. On the basis of these results, the non-toxic dose of ecabapide was considered to be 50 mg/kg in dogs and 100 mg/kg in rats.

Glucose-6-phosphate dehydrogenase (G6PD) screening and Heinz body preparation are both negative, and the haptoglobin level is decreased at 2.2 mg/dl (normal range: 27–139 mg/dl). C3 and C4 complement levels are normal, and the antinuclear antibody titer, anti-streptolysin O titer and Coombs tests are all negative. The paucity of intact red blood cells and the presence of pigment casts in the urinary sediment in this dark reddish-brown urine suggest the presence of hemoglobin or myoglobin. The moderate anemia, elevated reticulocyte count, and decreased haptoglobin are consistent with intravascular hemolysis. There are autoimmune causes of hemolytic anemia and G6PD deficiency can cause hemolytic anemia, but the Coombs test and G6PD preparation are normal. The fact that more than one hematopoietic cell line is abnormal suggests a clonal disorder. The history of mild leukopenia, thrombocytopenia, and hemolysis is consistent with paroxysmal nocturnal hemoglobinuria. The presence of a cell population lacking decay-accelerating factor (DAF) on the cell membrane determined by flow cytometric analysis with anti-DAF fluorescent antibody confirms the diagnosis.

Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

Arsine, the hydride of arsenic (AsH3), is the most acutely toxic form of arsenic, causing rapid and severe hemolysis upon exposure. The mechanism of action is not known, and there are few detailed investigations of the toxicity in a controlled system. To examine arsine hemolysis and understand the importance of various toxic responses, human erythrocytes were incubated with arsine in vitro, and markers of toxicity were determined as a function of time. The earliest indicators of damage were changes in sodium and potassium levels. Within 5 min incubation with 1 mm arsine, the cells lost volume control, manifested by leakage of potassium, influx of sodium, and increases in hematocrit. Arsine did not, however, significantly alter ATP levels nor inhibit ATPases. These changes were followed by profound disturbances in membrane ultrastructure (examined by light and electron microscopy). By 10 min, significant numbers of damaged cells formed, and their numbers increased over time. These events preceded hemolysis, which was not significant until 30 min. It has been proposed that arsine interacts with hemoglobin to form toxic hemoglobin oxidation products, and this was also investigated as a potential cause of hemolysis. Essentially on contact with arsine, methemoglobin was formed but only reached 2-3% of the total cellular hemoglobin and remained unchanged for up to 90 min. There was no evidence that further oxidation products (hemin and Heinz bodies) were formed in this system. Based on these observations, hemolysis appears to be dependent on membrane disruption by a mechanism other than hemoglobin oxidation.

Free radicals and reactive oxygen species have been implicated in the pathogenesis of a variety of hematologic diseases and erythrocyte aging. Aged erythrocytes are removed from the circulation primarily by the spleen. In this study, we aimed to determine the functional effectiveness of autotransplanted splenic tissue by its capacity to remove oxidatively modified erythrocytes from the circulation. Our experimental model in rats includes splenectomy with autotransplantation of 80% of the excised splenic tissue into the omental pouch. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as carbonyl content and thiobarbituric acid reactive substances (TBARS), together with Heinz body formation. Our results have shown that splenic autotransplantation was effective in removing oxidatively modified, aged erythrocytes from the circulation.

Onion (Allium cepa) toxicosis was diagnosed in a flock of local breed sheep, which had grazed daily on onion fields for 1 month. Two ewes died, four aborted and many showed clinical signs of toxicosis including pale mucous membranes, weakness and poor appetite. Haematology of 12 ewes revealed low PCV, erythrocyte number and haemoglobin, Heinz bodies in erythrocytes, polychromasia and mild leukocytosis. In those animals that died, there was haemoglobinuric nephrosis with haemosiderin deposition in tubular cells and centrilobular necrosis of liver. Onion feeding was discontinued and haematinics were used for treatment of the affected animals.

A 5-year-old pit bull-type dog was examined because of lethargy and black feces, and a presumptive diagnosis of methemoglobinemia was made on the basis of grossly visible brown discoloration of the mucous membranes and urine. Heinz body formation was seen on examination of blood smears, and severe anemia developed 3 days later. The dog did not have any history of exposure to toxins or drugs that have been associated with Heinz body formation. The only remarkable historical finding was that the dog had been wandering loose the day prior to initial examination and returned home smelling strongly of skunk spray. Skunk spray contains thiols and other compounds that are strong oxidizing agents. It was speculated that the methemoglobinemia and Heinz body anemia were a result of exposure to these compounds. Skunk interactions with dogs usually have malodorous but otherwise harmless results. However, findings in this dog suggest that more serious consequences may develop in isolated cases.

Background A captive Red Panda developed a regenerative anemia with Heinz bodies after being sprayed by a skunk. A definite cause-and-effect relationship between skunk musk and oxidative erythrocyte damage has not been reported, but it was suspected in one reported case of a dog with Heinz body hemolytic anemia. Objective The objective was to determine whether skunk musk induces oxidative HGB damage in vitro. Methods Plasma and RBC were harvested from heparinized blood of 3 dogs, 3 cats, and a Red Panda. Skunk musk was solubilized in ethanol and mixed with plasma from each species to make stock solutions of 4% musk and 4% ethanol. Aliquots of RBC were resuspended in autologous stock solutions and solvent controls to yield musk concentrations of 0%, 0.04%, and 0.4% (by volume). Aliquots were incubated at 37°C for 4–72 hours and assessed for oxidative damage by visual inspection, optical absorbance spectroscopy, transmission electron microscopy, and light microscopy after Wright and vital New Methylene Blue staining. Results Dose-dependent brown color and absorption changes characteristic of methemoglobin were present by 4 hours and increased over 24 hours (Red Panda) and 72 hours (dog and cat). Similarly, there were time-dependent (all species) and dose-dependent (dog and cat) increases in the number of Heinz bodies, which were present by 4 hours and numerous by 24 hours. Conclusions In vitro, skunk musk causes Heinz body and methemoglobin formation in canine, feline, and Red Panda RBC, supporting the clinical association between Heinz body hemolytic anemia and skunk spray exposure.

Because of the potential for exposure to N, N-dimethyl- p-toluidine (DMPT) in medical devices and the lack of toxicity and carcinogenicity information available in the literature, the National Toxicology Program conducted toxicity and carcinogenicity studies of DMPT in male and female F344/N rats and B6C3F1/N mice. In these studies, a treatment-related macrocytic regenerative anemia characterized by increased levels of methemoglobin and Heinz body formation developed within a few weeks of DMPT exposure in rats and mice. DMPT induced nasal cavity, splenic, and liver toxicity in rats and mice at 3 months and 2 years. DMPT carcinogenic effects were seen in the liver of male and female rats and mice, the nasal cavity of male and female rats, and the lung and forestomach of female mice. In rodents, DMPT is distributed to many of the sites where toxic and carcinogenic effects occurred. DMPT-induced oxidative damage at these target sites may be one mechanism for the treatment-related lesions. Methemoglobinemia, as seen in these DMPT studies, is caused by oxidation of the heme moiety, and this end point served as an early alert for other target organ toxicities and carcinogenic responses that followed with longer term exposure.

IZ Isaac,1 AS Mainasara,2 Erhabor Osaro,1 ST Omojuyigbe,1 MK Dallatu,3 LS Bilbis,3 TC Adias4 1Department of Haematology and Transfusion Medicine, 2Department of Chemical Pathology, 3Department of Biochemistry, Usmanu Danfodiyo University, Sokoto, Nigeria; 4Bayelsa State College of Health Technology, Ogbia, Nigeria Abstract: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human enzyme deficiencies in the world. It is particularly common in populations living in malaria-endemic areas, affecting more than 400 million people worldwide. This present study was conducted with the aim of determining the prevalence of G6PD deficiency among children visiting the Emergency Paediatric Unit of Usmanu Danfodiyo University Teaching Hospital for pediatric-related care. The study included 118 children, made up of 77 (65.3%) males and 41 (34.7%) females aged ≤5 years with mean age of 3.26 ± 1.90 years. Randox G6PD quantitative in vitro test screening was used for the diagnosis of G6PD deficiency. Of the 118 children tested, 17 (14.4%) were G6PD-deficient. Prevalence of G6PD deficiency was concentrated predominantly among male children (22.1%). Male sex was significantly correlated with G6PD deficiency among the children studied (r = 7.85, P = 0.01). The highest prevalence occurred among children in the 2- to 5-year age-group. Of the 17 G6PD-deficient children, twelve (70.2%) were moderately deficient, while five (29.4%) were severely deficient. Blood film from G6PD-deficient children indicated the following morphological changes; Heinz bodies, schistocytes, target cells, nucleated red cells, spherocytes, and polychromasia. This present study has shown a high prevalence of G6PD deficiency among children residing in Sokoto in the northwestern geopolitical zone of Nigeria. The study indicated a male sex bias in the prevalence of G6PD deficiency among the children studied. There is a need for the routine screening of children for G6PD deficiency in our environment, to allow for evidence-based management of these children and to ensure the avoidance of food, drugs, and infective agents that can potentially predispose these children to oxidative stress as well as diseases that deplete micronutrients that protect against oxidative stress. There is need to build capacity in our setting among pediatricians to ensure the effective management of children with G6PD deficiency. Keywords: G6PD, children, emergency pediatric unit, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria