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A 4-month-old male indoor cat was referred for dyspnea, mental dullness and weakness, which appeared two days earlier. The cat had been adopted at 3 months of age. Physical exam showed cyanosis, dyspnea and mild hypothermia. The “spot test” was supportive of methemoglobinemia. Co-oximetry blood gas analysis revealed severe methemoglobinemia (81.40%), severe hyperchloremia and mild hyponatremia. CBC, biochemistry and urinalysis were within normal limits, blood smear showed the presence of Heinz bodies. Treatment was instituted with oxygen therapy, methylene blue 1% solution, ascorbic acid, intravenous fluid therapy. The clinical course was favorable with rapid improvement of cyanosis and methemoglobinemia (4.2%). Acquired methemoglobinemia was hypothesized. Two weeks after discharge the cat was asymptomatic but mild methemoglobinemia (15.60%) and hyperchloremia were evident. Exposure to oxidants contained in drinking water was suspected so the owners were instructed to use bottled water only. One month later the cat was asymptomatic, and methemoglobinemia and chloremia were within normal limits. Even if a congenital form due to cytochrome b5 reductase deficiency cannot be ruled out, drinking water intoxication is the most likely cause of methemoglobinemia in this cat.

Background Plants of Allium spp., including garlic (A. sativum) and onions (A. cepa), are known to be oxidatively toxic to canine erythrocytes resulting in Heinz body hemolytic anemia in dogs. In humans, these plants have been used as medicinal agents for multiple diseases since ancient times. Especially, fresh garlic extracted over a prolonged period produces less irritative and odorless aged garlic extract (AGE), containing unique and beneficial organosulfur compounds that can help prevent many kinds of diseases. In this study, the safety and efficacy of long-term oral administration of AGE is evaluated in dogs. The objectives are to confirm the safe dosage for long-term use and beneficial functions of AGE for dogs and to plan and design a canine health supplement or a preventive agent for multiple diseases based on the data of this study. Results Beagles were orally administered AGE (45 or 90 mg/kg body weight once a day) or an equivalent amount of water as control for 12 weeks. In AGE-treated groups, at 12 weeks post-administration at a dose of 90 mg/kg, there were no observable changes in the clinical signs, complete blood count, and serum biochemical parameters. Heinz bodies and eccentrocytes, the markers of oxidative damage in erythrocytes, did not appear in blood smear examination. In order to further evaluate the beneficial effects of AGE on health of dogs, the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) gene (NFE2L2) and Nrf2-regulated phase II antioxidant enzyme genes (NQO1, GCLM, HMOX1, and SOD2) were determined in whole blood between pre- and post-AGE administration. The expression of NFE2L2 gene was significantly upregulated in the AGE-treated groups [45 (p

Heinz body formation has been reported in cats repeatedly administered propofol for anesthesia induction, although the resultant changes were deemed of little clinical significance (1, 2). This report suggests repeated propofol administration to some individual cats might induce anemia with clinical signs and cessation of propofol administration may result in rapid resolution. A 9-years-old American Domestic Shorthair cat receiving a 20-fraction radiation protocol for lateral thoracic fibrosarcoma showed lethargy, decreased appetite and activity, and Heinz body (3+ on blood smear examination) anemia (packed cell volume 22%; reference interval 24–45%) after 12 repeated propofol anesthesia inductions. The anesthesia induction protocol was adjusted to exclude propofol. Over the following week, the anemia resolved (packed cell volume, 30%), and the cat's activity level, appetite and attitude improved. The total dose of propofol received over the 12 treatments was 62.4 mg/kg.

International audience; Hb Dompierre [β29(B11)Gly→Arg, HBB: c.88G>C] is a rare β-globin gene variant that was previously described in the heterozygous state in a 24-year-old female patient. It is defined in the HbVar database as being clinically and biologically asymptomatic. A few years after the first description, we had an opportunity of reassessing the index case because she presented with splenomegaly and clinical and biological manifestations of hemolysis. After ruling out the most common causes of hemolysis, further analyses on the variant hemoglobin (Hb) using brilliant cresyl blue staining, indicated that it showed mild instability, which may explain the clinical and biological manifestations. A structural bioinformatic analysis on the Hb variant suggested that the amino acid replacement may be deleterious to the integrity of the Hb. This report confirms the importance of completely characterizing all new Hb variants in order to guide the patients' clinical management and follow-up, as well as to provide the probands and their family members with appropriate genetic counseling.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is known to cause haemolysis in patients with Hansen's disease receiving dapsone containing multidrug-therapy (MDT). However, sparse recent data are available on this topic from India. Methods: All patients aged over 10 years with Hansen's disease receiving MDT who presented with clinical deterioration to our tertiary care teaching hospital in Moradabad, Uttar Pradesh, India, were included in the study. G6PD levels were estimated in all of them. Once G6PD deficiency was confirmed dapsone was stopped and rescue therapy was initiated. Results: Between March 2015 and June 2016, 50 patients (mean age 34 ± 13.3 years) were included. G6PD deficiency was found in 14.3% patients. Peripheral blood smear showed Heinz bodies and bite cells in all of them. Mean pretreatment G6PD levels were 19.5 (range 17.5 – 25) units/g haemoglobin, mean serum bilirubin was 3.3 (range 1.6-9.2) mg/dL. Nineteen patients had lepra reactions (type 1 = 5 type 2 = 14). Ten patients required packed red blood cell transfusion. Conclusions: Our observations suggest that checking for G6PD levels before initiating dapsone containing MDT can be helpful in reducing the occurrence of haemolytic complications.

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.

Zinc toxicosis is commonly associated with ingestion of metallic zinc objects. This report documents an uncommon presentation of zinc toxicosis, secondary to prolonged ingestion of a zinc oxide cream. A 5.6 kg 6-year-old ovariohysterectomised female Poodle cross presented as a transfer to an emergency practice for severe anaemia, pigmenturia, weakness, and inappetence. She had a history of vomiting and nine days of diarrhoea. Amongst other supportive treatments, a zinc oxide cream had been applied to her hind end due to severe diarrhoea scalding, and in hindsight, the owners realised she had ingested large quantities of this cream. She developed a severe Heinz body haemolytic anaemia, along with spherocytosis, left-shift neutrophilia, prolonged activated partial thromboplastin time, and mildly elevated blood urea nitrogen. The serum zinc concentration was markedly elevated. She was treated supportively and made a full recovery. This case illustrates the importance of a thorough medical history. Zinc toxicosis can have a good prognosis when diagnosed and treated promptly.Keywords: Canine, Haemolytic anaemia, Heinz bodies, Toxicosis, Zinc

Lacquer thinner, commonly used for removing household paints, is known to contain various aromatic hydrocarbons and naphtha; if ingested, may cause methaemoglobinemia. We report a case of 13-year-old girl who presented with episodes of vomiting, abdominal pain and numbness of limbs. Peripheral blood smear showed few fragmented erythrocytes and 10-12 nucleated red blood cells /100 white blood cells. Reticulocyte count was 15% with presence of Heinz bodies. There was a history of accidental ingestion of lacquer paint thinner. Levels of methaemoglobin were very high along with raised HbA2 levels. An impression of haemolytic anaemia, due to lacquer thinner paint poisoning, with methaemoglobinemia was given. Incidentally High Performance Liquid Chromatography (HPLC) revealed increased HbA2 levels i.e., heterozygous inheritance of beta thalassaemia minor. Patient responded well to methylene blue. Blood transfusion was performed and symptomatic treatment was given.

BACKGROUND: Antibiotic prophylaxis against Pneumocystis jirovecii pneumonia (PCP) is recommended for glioblastoma (GBM) patients receiving radiation and temozolomide. Trimethoprim-sulfamethoxazole (TMP-SMX), dapsone, atovaquone, and inhaled pentamadine are accepted antibiotics. Oxidant hemolysis is a known complication of dapsone, typically associated with G6PD deficiency. We report hemolytic anemia in 7/12 GBM patients treated with dapsone. We compare the rate of anemia with TMP-SMX and pentamadine-treated patients. METHODS: Retrospective review of charts of GBM patients treated with radiation and temozolomide 2009 -14 at our institution. RESULTS: Of 52 patients, 23 received TMP-SMX, 17 pentamadine, and 12 dapsone. Seven (58%) of the dapsone treated patients experienced > 2.0 g/dl drop in hemoglobin from baseline. In each patient, G6PD screening was normal. Macrocytosis was present in 4/7 dapsone-treated patients with anemia. Lactate dehydrogenase levels were elevated (range 312-414 U/L) in 3 / 4 in whom it was available. Peripheral smears of 5 demonstrated degmacytes (‘bite cells’). CONCLUSION: Anemia was more common in patients treated with dapsone than with TMP-SMX or pentamadine. The frequency of anemia is higher than anticipated, especially with normal G6PD levels. Evidence of hemolysis is supported by characteristic bite cells, increased LDH levels, with compensatory reticulocytosis suggested by macrocytosis. Close monitoring for anemia in GBM patients treated with dapsone for PCP prophylaxis is required, even with normal pretreatment G6PD levels.

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.

In order to investigate daily changes of hematological parameters in bleeding-induced anemia, we treated Wistar albino male rats by daily bleeding (1.5-2 mL of blood from the tail vein for eight days). Blood samples were taken before (on day zero) and on the first to eighth days of bleeding. The values of hematocrit, hemoglobin, and erythrocyte count decreased significantly after the second, sixth, and second days of bleeding, respectively. The number of leuko?cytes and platelets, as well as Heinz body levels, increased significantly after the third and second days of treatment. The percentage of reticulocytes increased significantly from the second day and attained the maximum level (32.55 ? 0.96%) on the eighth day.

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

During the four month period, from December 1988 to March 1989, there was an outbreak of Heinz body positive hemolytic anemia in 34 patients undergoing hemodialysis in a 500-bed hospital, Seoul, Korea. The episodes of hemolysis were not reduced by changing the charcoal column and reverse osmosis system, or by adding ascorbic acid to the dialysate. The concentrations of nitrate, copper, aluminum and zinc in the treated water were all within the standards for hemodialysis. The chloramine concentration of the treated water was over 0.6 mg/L, markedly exceeding the allowable level of 0.1 mg/L. This high level of chloramine was proved to be due to the contamination of the water source by raw sewage. After we changed the source of water supply to another, no more episodes of hemolytic anemia occurred. It is concluded that chloramine is one of the major contaminants causing dialysis-induced hemolytic anemia and regular determinations are necessary, especially during winter and dry seasons.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:47Z No. of bitstreams: 1 S1415-47572000000200020.pdf: 458000 bytes, checksum: 59b543dcffadfe57b299da36c89614bc (MD5) Made available in DSpace on 2013-08-22T19:04:47Z (GMT). No. of bitstreams: 1 S1415-47572000000200020.pdf: 458000 bytes, checksum: 59b543dcffadfe57b299da36c89614bc (MD5) Previous issue date: 2000-06-01 Made available in DSpace on 2013-09-30T20:08:07Z (GMT). No. of bitstreams: 2 S1415-47572000000200020.pdf: 458000 bytes, checksum: 59b543dcffadfe57b299da36c89614bc (MD5) S1415-47572000000200020.pdf.txt: 28143 bytes, checksum: 3b999ca559c0c81fe38b54ea45a06050 (MD5) Previous issue date: 2000-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:17:24Z No. of bitstreams: 2 S1415-47572000000200020.pdf: 458000 bytes, checksum: 59b543dcffadfe57b299da36c89614bc (MD5) S1415-47572000000200020.pdf.txt: 28143 bytes, checksum: 3b999ca559c0c81fe38b54ea45a06050 (MD5) Made available in DSpace on 2014-05-20T15:17:24Z (GMT). No. of bitstreams: 2 S1415-47572000000200020.pdf: 458000 bytes, checksum: 59b543dcffadfe57b299da36c89614bc (MD5) S1415-47572000000200020.pdf.txt: 28143 bytes, checksum: 3b999ca559c0c81fe38b54ea45a06050 (MD5) Previous issue date: 2000-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A eritropoese e a atividade de transcrição de DNA de hemoglobina foram analisadas em Odontophrynus americanus diplóides e tetraplóides. Dados de conteúdo celular relativo de DNA, RNA e mitocôndrias obtidos por citometria de fluxo mostraram uma atividade gênica aumentada em ambos os animais durante a eritropoese in vivo estimulada por tratamento com fenilhidrazina, com valores maiores em eritrócitos tetraplóides imaturos. No décimo dia de recuperação da anemia foram encontradas grandes quantidades de RNA mensageiro em ambos espécimens. Com base no conteúdo mitocondrial, as células tetraplóides apresentaram um metabolismo energético mais intenso. O. americanus diplóides apresentaram três vezes mais células eritróides que os animais tetraplóides, caracterizando um mecanismo regulador da produção de células eritróides. Determinações hematológicas mostraram que as células tetraplóides contêm 30% mais hemoglobina que as células diplóides. Uma vez que 25-30% mais ribossomos estão presentes nas células tetraplóides, parece que um mecanismo regulador da síntese de hemoglobina esteja relacionado ao nível transcricional. Como as inclusões citoplasmáticas semelhantes a corpos de Heinz, porém associadas com RNA ou RNP, são encontradas em ambos os espécimens, é possível que outro mecanismo regulador esteja presente, similar ao das hidden breaks descritas em peixes poliplóides, acumulando rRNA transcrito em excesso e não traduzido. Estas diferenças em níveis fisiológicos e moleculares, detectadas durante a eritropoese, reforçam que divergências em nível de especiação estão em curso entre os O. americanus diplóides e tetraplóides. Erythropoietic and hemoglobin DNA transcriptional activities were analyzed in the diploid and the tetraploid Odontophrynus americanus. Flow cytometric analyses of DNA, RNA and mitochondrial contents showed increased genic activity in both diploid and tetraploid animals during erythropoiesis in vivo elicited by pretreatment phenylhydrazine. Generally, higher values were seen in immature tetraploid erythroid cells. on the 10th day of recovery from anemia, large amounts of messenger RNA were found in both specimens. Based on the mitochondrial content, the tetraploid cells had more intense energy metabolism than the diploid cells. Diploid O. americanus had about three times more erythroid cells than tetraploid specimens, indicating that there were differences in the regulatory mechanisms of erythroid cells. Hematological parameters showed that tetraploid cells had 30% more hemoglobin than the diploid, suggesting a regulatory mechanism of hemoglobin synthesis at the transcriptional level. Cytoplasmic inclusions resembling Heinz bodies were found in both types of cells. In the tetraploid cells they were previously found associated with RNA or RNP, suggesting that other regulatory system which controls the accumulation of nontranslated RNA transcribed in excess must be present. These differences at the physiological and molecular levels during erythropoiesis reinforce the hypothesis that speciation is occurring between diploid and tetraploid O. americanus. Instituto Butantan Laboratório de Genética Universidade Estadual Paulista Departamento de Biologia Laboratório de Hemoglobinas e Hemoglobinopatias Fundação Oswaldo Cruz Departamento de Protozoologia Fundação Oswaldo Cruz Departamento deUltra-estrutura e Biologia Celular Universidade Estadual Paulista Departamento de Biologia Laboratório de Hemoglobinas e Hemoglobinopatias

Background Sickle cell anemia is one of the most prevalent hereditary disorders with prominent morbidity and mortality. With this disorder oxidative, phenomena play a significant role in its pathophysiology. One of the garlic (Allium sativum L.) formulations, aged garlic extract (AGE), has been reported to exert an anti-oxidant effect in vitro, we have evaluated the anti-oxidant effect of AGE on sickle red blood cells (RBC). Methods Five patients (two men and three women, mean age 40 ± 15 years, range 24–58 years) with sickle cell anemia participated in the study. AGE was administered at a dose of 5 ml a day. Whole blood samples were obtained at baseline and at 4 weeks for primarily Heinz body analysis. Results The data were consistent with our hypothesis. In all patients, the number of Heinz bodies decreased over the 4 week period (58.9 ± 20.0% at baseline to 29.8 ± 15.3% at follow-up, p = 0.03). Conclusions These data suggest that there is a significant anti-oxidant activity of AGE on sickle RBC. AGE may be further evaluated as a potential therapeutic agent to ameliorate complications of sickle cell anemia.

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.

Hemoglobin Mississippi (HbMS: beta 44ser----cys) has anomalous properties that include disulfide linkages with normal beta-, delta-, gamma-, and alpha-chains, and the formation of high molecular weight multimers. While heterozygotes for HbMS are clinically and hematologically normal and carriers of the beta +-thalassemia gene in our family had mild microcytic anemia, the proband with HbMS-beta +-thalassemia had a hemoglobin level of 7 g/dl, mean corpuscular volume (MCV) of 68 fl, reticulocytes of 2-6%, HbF of 18%, marked anisocytosis and poikilocytosis, and splenomegaly, all features of thalassemia intermedia. With oxidant stress, her erythrocytes developed multiple dispersed Heinz bodies, but HbMS was only mildly unstable. HbMS was susceptible to proteolytic degradation in the presence of ATP. The unexpectedly severe clinical findings in HbMS-beta +-thalassemia may result from the proteolytic digestion of HbMS, as well as the excessive alpha-chains characteristic of beta +-thalassemia, which combined provide the increment of cellular damage that results in the phenotype of thalassemia intermedia.

Of 52 patients receiving a mean dose of 2.5g sulphasalazine/day as maintenance therapy for ulcerative colitis, 35 were found to have one or more drug-induced red cell abnormalities, which were not found in 50 normal controls or in 10 colitics not receiving sulphasalazine. Twenty-three of the treated patients had contracted red cells, an abnormality that is thought to result in mild haemolysis. Red cell contraction was related to the dose of sulphasalazine (P smaller than 0.01), the serum total sulphapyridine level (P smaller than 0.001), and acetylator status. Eleven of the treated patients had a macrocytosis, 21 had elevated levels of methaemoglobin, and one had Heinz bodies. A dose of 1.5 g sulphasalazine/day was not associated with red cell contraction, and is suggested as a safer maintenance dose for the asymptomatic colitic.

Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.

1 The relationship between mechanical fragility, glutathione peroxidase inhibition and Heinz body formation, in erythrocytes exposed to oxidant drugs in vitro, has been investigated. All drugs tested caused Heinz body formation, and with the exception of acetyl salicylic acid and salicylic acid, also caused increased erythrocyte mechanical fragility. 2 There was a direct relationship between mechanical fragility and drug concentration. Mechanical fragility increased in parallel with Heinz body formation, with primaquine, gentisic acid, ascorbic acid and potassium chlorate. In contrast Heinz body formation occurred at drug concentrations which did not cause a marked increase in mechanical fragility in the case of menadione, acetyl phenylhydrazine and phenylhydrazine. 3 The degree of inhibition of glutathione peroxidase was directly related to increased mechanical fragility with menadione, gentisic acid and potassium chlorate. However other substances causing increased mechanical fragility resulted in little or no loss of glutathione peroxidase activity. 4 The results show that there is no constant relationship between mechanical fragility caused by drugs, the formation of Heinz bodies and the inhibition of glutathione peroxidase. The factors contributing to oxidant drug-induced haemolysis appear to be variable and depend upon the drug concerned.

Three patients developed autoimmune haemolytic anaemia while being treated with mefenamic acid. In each case the autoimmune haemolytic anaemia was of the warm antibody γG type, and the antibodies had some rhesus specificity. All three patients recovered when the drug was withdrawn. Attempts to inhibit or enhance the activity of the antibody in vitro were unsuccessful. Direct antihuman globulin tests were made in.the red cells of 36 patients receiving long-term mefenamic acid therapy, but only one was found to be transitorily positive.