Heinz body-related interference with leukocyte and erythrocyte variables obtained by an automated hematology analyzer in cats.

Heinz bodies (HBs) are known to interfere with automated hematology in cats, particularly with the white blood cell (WBC) count. We evaluated the influence of feline HBs on the complete blood count (CBC) results obtained using a flow cytometry-based analyzer. We retrospectively selected cats with circulating HBs and reviewed the results of their CBCs, including red blood cell (RBC) indices, basophil/lobularity (Baso) WBC count (WBCB), peroxidase (Perox) WBC count (WBCP), and cytograms. Based on the presence or absence of HB-related artifacts in their Baso cytogram, cats were grouped into Baso-HBs and HBs groups, respectively, for comparison. The WBCB and WBCP were compared to manual counts of WBCs carried out on blood smears at 400× (MC-WBC). We included 32 cats in our study: 9 of 32 were in the Baso-HBs group, and 23 of 32 were in the HBs group. Baso-HBs cats had a significantly increased HB percentage (p < 0.001), WBCB (p < 0.001), difference between WBCB and WBCP (p < 0.001), lymphocyte count (p < 0.001), mean corpuscular hemoglobin concentration (p < 0.001), and difference between calculated and measured erythrocyte hemoglobin concentrations (p < 0.001) compared to HBs cats. In Baso-HBs cats, the WBCB was significantly higher than the WBCP (p = 0.02); no significant difference was detected between the WBCP and the MC-WBC (p = 0.88). Evaluation of automated CBC results raised the suspicion of HB-related interference when using a hematology analyzer in cats; hence, blood smear examination remains essential in routine practice.

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

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

Plasma-Saturating intakes of vitamin C confer maximal antioxidant protection to plasma.

OBJECTIVE: Supplemental vitamin C has been shown to reduce oxidative damage in vivo, yet the dose-response relationship between vitamin C intake and antioxidant protection is not known. This report examined blood indicators of oxidative stress in subjects consuming graded doses of vitamin C, from 75 to 2,000 mg/day. METHODS: Ten healthy, non-smoking men and women (26.1 +/- 2.1 years) were recruited from a campus population. During the ten-week study, subjects limited fruit and vegetable consumption (< or = 3 servings/day) and consumed a multivitamin and mineral pill daily. Beginning at week 3, subjects ingested either a vitamin C (n = 8) or placebo (n = 2) capsule, which were identical in appearance and taste. The content of the vitamin C capsule increased every two weeks (from 250 mg at weeks 3-4 to 500 mg, 1,000 mg. and 2,000 mg at weeks 5-6. 7-8. 9-10, respectively). Fasting blood samples were collected at two-week intervals and analyzed for vitamin C, total lipid hydroperoxides and Heinz bodies in packed erythrocytes incubated 24 hours at 37 degrees. RESULTS: Plasma vitamin C rose 55% in vitamin C-supplemented subjects by the end of the ten-week treatment (p < 0.05), and measures of oxidative stress decreased 60% to 90% (8.1 +/- 0.6 to 3.5 +/- 0.4 nmol/mL and 69.1 +/- 7.8% to 6.7 +/- 6.0% for total lipid hydroperoxides and Heinz bodies, respectively). Significant decreases in markers of oxidative stress were noted at the 500 mg, 1,000 mg and 2,000 mg dosages versus placebo. Antioxidant protection was similar at the 1,000 mg and 2,000 mg dosage. CONCLUSIONS: These data indicate that the antioxidant protection afforded by short-term vitamin C supplementation is maximal at the 500-1,000 mg dosage range.

[Mechanisms for the enhanced red cell destruction in hemoglobinopathy].

The process of denaturation of Hb within red cells, leading to ultimate formation of Heinz body or aggregates of irreversibly denatured Hb, was formulated over 20 years ago. The mechanical obstruction due to Heinz bodies seems self-evident as a cause of enhanced red cell destruction. Recent studies compare the overall defects in rheologic properties of red cells of unstable Hb disorder with artificially oxidized red cells. The fate of superoxide anion which is the other product of oxidative denaturation of Hb is being discussed in the light of accumulation of oxidative damage to red cell membrane. From this aspect, unstable Hb disorder may be regarded as a model of accelerated red cell aging. The aged red cells appear to bind increased amounts of immunoglobulin. They may express scavenger ligands. The rheologic defects and altered surface phenomena would be additive in enhancing red cell phagocytosis. The chemical basis for the abnormal properties of unstable Hb red cells, as well as normal senescent red cells, is yet to be defined.

Rheologic and pathophysiologic significance of red cell passage through narrow pores.

To elucidate the pathophysiologic significance of red blood cell (RBC) filterability, we measured RBC rheology with our own designed nickel mesh with 3-microns pores, smaller than the previously used 5-microns pores. Vertical and cylindrical pores with no pore coincidence were regularly distributed across the filter, the pore entrances of which showed a round and rather smooth transition to the pore inside. An advantage of the nickel mesh is the repeated use (at least 100 times) of the same filter possible after ultrasonic washing. A very low concentration of RBC, i.e., 3 x 10(4) cells per cubic millimeter (hematocrit value of approximately 0.3%), was sufficient for a typical test to examine RBC filterability. The filtration of the dilute RBC suspension was not influenced by contaminating or added leukocytes up to a leukocyte count of approximately seven cells per cubic millimeter; therefore, measurements can be performed using conventionally washed RBCs. This may be practically relevant to routine use, such as in a clinical laboratory. As compared with filtration through 5-micron pores, filtration through 3-micron pores was found to be very sensitive in detecting major determinants of RBC deformability, particularly, changes in viscoelastic properties of the cell membrane, surface area/volume ratio of the cell, perturbing effects of lysophosphatidylcholine, and osmolality of the medium. The 3-micron filtration method revealed a marked impairment in the filterability of Heinz body-containing RBCs from patients with unstable hemoglobin (Hb) disease (Hb Yokohama). Thus, 3-micron-filtration measurements may contribute to several subfields of hematology.

Relation between reduced glutathione content and Heinz body formation in sheep erythrocytes.

To clarify the oxidant defense functions of reduced glutathione (GSH) in erythrocytes, the effect of GSH deficiency on in vitro oxidant defense was studied, using GSH-deficient sheep erythrocytes (low-GSH cells). The formation of Heinz bodies in low-GSH cells was higher than that in high-GSH cells when the cells were incubated with an oxidant drug, acetylphenylhydrazine (APH). Artificial depletion of GSH by 1-chloro-2,4-dinitrobenzene in high-GSH cells resulted in increased Heinz body formation in these cells incubated with APH. Furthermore, high negative correlation was observed between Heinz body formation and GSH content in sheep erythrocytes exposed to APH. These results clearly indicate that erythrocyte GSH is indispensable for erythrocyte defense against oxidative damage induced by APH, and support the previous observations that sheep with low-GSH erythrocytes were more susceptible to oxidative agents than were sheep with high-GSH erythrocytes.

Erythrocyte membrane deformability in patients with thalassemia syndromes.

The purpose of this study was to measure the erythrocyte membrane's elasticity in patients with thalassemia syndromes. These syndromes have been well investigated regarding the problems associated with the hemoglobin solution. A few studies dealing with the whole cells' flexibility have also been published. However, there are extremely few studies regarding the elastic behaviour of the membrane of these cells which plays a significant role in the viability of these erythrocytes especially when they transverse the small capillaries of the spleen. The micropipette aspiration method was used for this purpose. Cells from patients with thalassemia syndromes were aspirated into micropipettes of internal diameter of 1.2-1.6 microns. The slope of the curve relating the length of the aspirated tongue to the negative pressure was measured to give the elastic shear modulus. This modulus was compared with the one of the normal cells. Inclusions (Heinz bodies) were produced artificially in normal cells and their elastic shear modulus was also measured. The results show that the elastic shear modulus measured in homozygous beta-thalassemia was 33% higher than the normal one. The elastic shear modulus in cells with artificial Heinz bodies was also higher than the normal one and its value increased with the number of the inclusion Heinz bodies. The elastic shear modulus in heterozygous beta-thalassemia was slightly higher than the normal one. The apparent stiffening of the membrane of the homozygous cells might be partially due to the "constraining effect" of the inclusions to the membrane's ability to deform freely.

On the mechanism of loss of deformability in human erythrocytes due to Heinz body formation: a flow EPR study.

The effects of phenylhydrazine (PHZ) and monomethylhydrazine (MMH) on the deformability of human erythrocytes and ghosts in relation to Heinz body formation are investigated using the sensitive flow EPR (electron paramagnetic resonance) method. The decrease in deformability starts even before Heinz body formation is recognized and is enhanced with PHZ as Heinz bodies are attached on the inner surface of the membrane, but remains at a low level in MMH-treated cells in which Heinz bodies are formed mostly away from the membrane surface. Dithioerythritol recovers part of the lost deformability by MMH but has no effect in PHZ-treated cells or in the ghosts. The membrane fluidity measurements by 12-doxyl stearate indicate that there are two distinct modes of spin-label interaction in PHZ-treated cell membrane, the one corresponding to more immobilization gaining with the PHZ concentration, while MMH has only minor effects on fluidity. The motional freedom of membrane proteins probed with maleimide label shows that the membrane binding of hemoglobin denatured with PHZ causes more immobilization than that with MMH. These observations and Fe(III) EPR absorptions of the treated cells and ghosts are interpreted on the basis of the difference in reaction products (denatured hemoglobin) by the two reagents: the ones with PHZ preferentially bind to the membrane proteins, such as band 3, resulting in a perturbation of the membrane viscoelastic properties, while MMH-denatured hemoglobins remain mostly in the cytosol as Heinz bodies, or in the polymeric form contributing to the internal viscosity.

Contribution of propylene glycol-induced Heinz body formation to anemia in cats.

Propylene glycol (PG) is a common preservative and source of synthetic carbohydrates in soft-moist pet foods. Propylene glycol was fed to cats for 5 weeks at concentrations found in commercial diets (1.6 g/kg of body weight; 12% of diet on a dry-weight basis) and for 3 weeks at concentrations exceeding usual intake (8 g/kg; 41% of diet). There was a dose-dependent increase in Heinz body percentage to 28% in cats fed the low dose of PG and to 92% in cats fed the high dose. Erythrocyte half-life, measured using [14C]-cyanate hemoglobin (Hb), decreased significantly (P less than 0.05) by 18.8% and 60% in cats fed the low and high PG doses, respectively. The PCV in cats fed the low dose was unaffected, whereas cats fed the high dose had a mean (+/- SEM) decrease in PCV from 33.5 +/- 1.05% to 26.3 +/- 1.45%, accompanied by punctate reticulocytosis and bone marrow erythroid hyperplasia. A dose-dependent increase in iron pigment was found in the liver and spleen of all cats. In cats fed the low dose of PG, erythrocyte reduced glutathione concentration actually increased from 7.02 +/- 0.56 to 9.74 +/- 0.69 mumol/g of Hb, but decreased to 2.96 +/- 0.27 mumol/g of Hb in cats fed the high dose. There was no significant increase in methemoglobin concentration. These results indicated that PG cannot be considered innocuous even at concentrations consumed by cats eating commercial diets. Heinz body-induced acceleration of RBC destruction develops in a dose-dependent manner, so that cats with greater food intake, ie, lactating queens and nursing kittens, are at greater risk for development of PG-induced Heinz body hemolytic anemia.

Heinz bodies induce clustering of band 3, glycophorin, and ankyrin in sickle cell erythrocytes.

In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.

Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia.

Temporal changes in the levels of denatured haemoglobin (Heinz bodies) and fluorescent lipid peroxidation products in the red cells of rabbits administered phenylhydrazine have been followed. Heinz bodies were maximal just before the period when most of the cell destruction occurred, whereas lipid peroxidation products were maximum when reticulocyte levels were highest. This implies that lipid peroxidation occurs mainly in immature cells and that haemoglobin denaturation is more likely than lipid peroxidation to be a major contributor to haemolysis.

Heinz body production and hematological changes in the hen after administration of a single oral dose of n-butyl mercaptan and n-butyl disulfide.

n-Butyl mercaptan (nBM) is a breakdown product of S,S,S,-tri-n-butyl phosphorotrithioate (DEF) and S,S,S-tri-n-butyl phosphorotrithioite (merphos) in hens and in the environment. n-Butyl disulfide (nBD) is an oxidation product of nBM. A single 500 mg/kg dose of nBM and nBD was administered in gelatin capsules to groups of five 12-month old laying hens. A third group (five hens) was given gelatin capsules. One day after administration, the hens exhibited weakness which progressed to unsteadiness and inability to stand by the third day. These signs were accompanied by a pale comb 18--24 hr after dosing, which changed to dark color at 48 hr. Treated hens improved with time. Heinz bodies and extensive erythrocyte deformation and lysis were observed in blood smears taken from hens 24 and 48 hr after treatment. Hemoglobin concentration, packed cell volume, erythrocytes, and glucose-6-phosphate dehydrogenase activity were significantly lower than controls, while methemoglobin was significantly higher. As the clinical condition of these hens improved, these hematologic changes disappeared. nBM caused an initial increase in plasma butyrylcholinesterase activity which was dose-dependent and returned to normal by the end of the 28-day experiment. Also, brain acetylcholinesterase activity was not different from that of the control at termination.

[Abnormal cation transport in erythrocyte membranes during congenital hemolytic anemia (author's transl)]. / Anomalies des transferts cationiques transmembranaires au cours des anémies hémolytiques congénitales.

In this paper we review our knowledge on: 1. The physiological mechanisms of constant low Na+, high K+ concentration in human red blood cells (cation content, cation movements through the erythrocyte membrane, membrane ATPases). 2. The pathologic findings observed in congenital hemolytic anemia. These alterations may explain hyperhemolysis in numerous diseases. In no case a primary genetically transmitted abnormal erythrocyte membrane protein can be formely demonstrated.