Methemoglobin is a supplement for in vitro culture of human nasopharyngeal epithelial cells transformed by human Papillomavirus type 16 DNA

NPC-N cells were normal human nasopharvngeal epithelial cells transformed by transfection with human papillomavirus type 16 deoxyribonucleic acid. Bovine pituitary extract (BPE) was one of the indispensable ingredients for in vitro culture of NPC-N cells in a serum-free medium. Chromatographic fractionation of BPE and subsequent immunoblotting analyses identified the hemoglobin growth-stimulating factor. Methemoglobin (metHb) was then synthesized, and also found to be growth stimulating. The growth-stimulating effect of metHb was abolished when NPC-N cells were cultured in a medium that also contained haptoglobin, a molecule that binds to hemoglobin. A defined medium consisting of insulin and metHb was then developed for optimal growth of NPC-N cells. MetHb kept under the conditions identical to those of cell culture released hemin which also enhanced the cell growth. Though all the degradation products of hemin are currently known to be physiologically significant. only ferric iron derived from metHb or hemin could stimulate the growth of NPC-N cells. Abnormal vasculature showing leaky walls and hemorrhage is a common feature of malignant tumors. Hemoglobin originating from extravasated red blood cells and subsequently oxidized to metHb because of the presence of activated inflammatory cells might contribute to the increased proliferation of cancerous cells.     

Potential of peroxynitrite to promote the conversion of oxyhemoglobin to methemoglobin

Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite (ONOO-)which can react directly with hemoglobin (Hb) even in the presence of physiological concentration CO:. Thisresearch was to determine the ONOO--mediated oxidation damage to the heme of oxyhemoglobin (oxyHb)under conditions expected in blood. Results showed that 8-10 mol ONOO- was needed to quickly andcompletely convert 1 mol oxyHb to methemoglobin (metHb). ONOO- (20-140 μM) caused raoid andextensive formation of metHb from oxyHb (50 μM) mainly occurring within first 5-20 min of incubation.The conversion efficiency reached 16%, 48%, 60%, 79% and 88% output of metHb after 90 min ofincubation at 0, 20, 40, 100, and 140 μM ONOO- respectively. 1 mM CO2 caused a small decrease in theability of ONOO- to oxidize oxyHb, and ONOO--promoted conversion of oxyHb to metHb increased whenpH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction insome degree. We postulate that ONOO- can mediate oxidation damage to the heme, and cause heme lossfrom the hydrophobic cavity of Hb when its concentration exceeded 90 μM. These results indicated thatONOO- could convert oxyHb to metHb under the conditions expected in blood, and this reaction wasregulated by CO2 concentration, reaction time, temperature and pH value.     

No scavenging and the hypertensive effect of hemoglobin-based blood substitutes

The major pathway for nitric oxide scavenging in red cells involves the direct reaction of the gas with HbO2 to form nitrate and the ferric form of the protein, metHb. Because both atoms of O2 are incorporated into nitrate, this process is called NO dioxygenation (NOD). The NOD reaction involves an initial, very rapid bimolecular addition of NO to bound O2 to form a transient Fe(III)-peroxynitrite complex, which can be observed spectrally at alkaline pH. This intermediate rapidly isomerizes at pH 7 (t1/2 <== 1 ms) to metHb and NO3-, which is nontoxic and readily transported out of red cells and excreted. The rate of NO consumption by intracellular HbO2 during normal blood flow is limited by diffusion up to and into the red cells and is too slow to interfere significantly with vasoregulation. In contrast, extracellular HbO2 is highly vasoconstrictive, and the resultant hypertension is a significant side effect of most hemoglobin-based blood substitutes. The major cause of this blood pressure effect seems to be the high rate of NO dioxygenation by cell-free HbO2, which can extravasate into the vessel walls and interfere directly with NO signaling between endothelial and smooth muscle cells. This interpretation is supported by a strong linear correlation between the magnitude of the blood pressure effect caused by infusion of cross-linked recombinant hemoglobin tetramers in vivo and the rate of NO dioxygenation by these proteins measured in vitro.     

Membrane effects of nitrite-induced oxidation of human red blood cells.

The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.     

Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular "free" heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation. Hence in man the regulatory process affects generation of the first committed precursor of porphyrin biosynthesis by delta-aminolevulinate synthetase, whereas in the rabbit separate regulatory mechanisms exist which control the incorporation of iron into protoporphyrin IX.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

Hydroxy-urea protects erythrocytes against oxidative damage

Hydroxy-urea (OH-U) is used to treat sickle cell anemia by increasing hemoglobin fetal-fraction. It has been suggested that the sickle cell mutations lead to the formation of unstable HbS and release of iron, which can result in lipid peroxidation (LPO), and eventual cell damage. Since oxidative processes might be involved in pathogenesis of sickle cell disease, we investigated the antioxidant property of OH-U in a red blood cell (RBC) model. Intact RBCs or RBC membranes were exposed to t-butyl hydroperoxide (t-BHP, 0.75 mM) or iron (ferrous sulfate; 100 microM) at 37 degrees C for 60 min in the presence or absence of OH-U (1.25 mM). The extent of oxidative damage was measured by LPO (as thiobarbituric acid reactive substances, TBARS), hemoglobin oxidation (as percent of methemoglobin, metHb %), and decrease in the activities of membrane-bound Na+/K+-ATPase and Ca2+-ATPases. Our results show that OH-U inhibited t-BHP-induced LPO in fresh RBC membranes significantly (P <0.01). OH-U significantly inhibited t-BHP-mediated LPO (P <0.01) and metHb formation (P <0.01) in intact RBC. Also, OH-U inhibited iron-induced LPO and metHb formation in intact RBC (P <0.01). In addition, OH-U blocked t-BHP-mediated changes in membrane ATPase activities. Furthermore, OH-U blocked iron-mediated hydroxyl radical generation in a dose-dependent fashion. In conclusion, the observed antioxidant properties of OH-U might contribute to its therapeutic action in sickle cell disease.     

Mechanism of peroxynitrite interaction with ferric hemoglobin and identification of nitrated tyrosine residues. CO(2) inhibits heme-catalyzed scavenging and isomerization.

Hemoproteins are one of the major targets of peroxynitrite in vivo. It has been proposed that the bimolecular heme/peroxynitrite interaction results in both peroxynitrite inactivation (scavenging) and catalysis of tyrosine nitration. In this study, we used spectroscopic techniques to analyze the reaction of peroxynitrite with human methemoglobin (metHb). Although conventional differential spectroscopy did not reveal heme changes, our results suggest that, in the absence of bicarbonate, the heme in metHb reacts bimolecularly with peroxynitrite but is quickly back-reduced by the reaction products. This hypothesis is based on two indirect observations. First, metHb prevents the peroxynitrite-mediated nitration of a target dipeptide, Ala-Tyr, and second, it promotes the isomerization of peroxynitrite to nitrate. Both the scavenging and the isomerization activities of metHb were heme-dependent and inhibited by CO(2). Ferrous cytochrome c was an efficient scavenger of peroxynitrite, but in the ferric form did not show either scavenging or isomerization activities. We found no evidence of an increase in Ala-Tyr nitration with these hemoproteins. Peroxynitrite-treated metHb induced the formation of a long-lived radical assigned to tyrosine by spin-trapping studies. This radical, however, did not allow us to predict an interaction of peroxynitrite with heme. Hb was nitrated by peroxynitrite/CO(2) mainly in tyrosines beta 130, alpha 42, and alpha 140 and, to a lesser extent, alpha 24. The nitration of alpha chain tyrosines more exposed to the solvent (alpha 140 and alpha 24) was higher in CO-Hb and metHb, while nitration of alpha 42, the tyrosine nearest to the heme, was higher in oxyHb. We deduce that the heme/peroxynitrite interaction, which is inhibited in CO-Hb and metHb, affects alpha tyrosine nitration in two opposite ways, i.e., by protecting exposed residues and by promoting nitration of the residue nearest to the heme. Conversely, nitration of beta Tyr 130 was comparable in oxyHb, metHb, and CO-Hb, suggesting a mechanism involving only nitrating species formed during peroxynitrite decay.     

Characteristics of hematin uptake in malignant, embryonic and normal cells

The characteristics of hematin uptake were examined in three malignant cell lines [L1210 leukemia, 745 murine erythroleukemia (MEL) and Walker carcinoma (W256)], a cell line derived from normal rat liver (BRL-3A) and a normal embryonic cell, chick embryo fibroblasts (CEF). Uptake in the normal liver cell line was slight and occurred at a slow rate in contrast to the rapid uptake, which was more rapid and of greater magnitude in the three tumor cell lines, Saturation of the heme uptake mechanism was observed in MEL cells at an extra-cellular hematin concentration of 160 micro M and in L1210 cells at 300 micro M. At saturation L1210 cells achieved a cellular heme concentration nine times as high as MEL cells. Hematin uptake in MEL cells was markedly augmented by pretreatment with DMSO, procaine, detergent or proteolytic enzymes or by increases in the pH of the medium from 8 to 9.5. In contrast to MEL cells where SA inhibits growth by lowering cellular heme, the inhibition of growth of L1210 cells by SA appears to operate by a mechanism independent of heme. In gradual increase in hematin uptake capacity in MEL cells over a period of days. Afer exposure of MEL cells to a high concentration of hematin in the medium, the egress of heme was followed under various conditions. Of the various agents studied, only cyanide produced a loss of heme from MEL cells.     

Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical

To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.     

Enhanced botulinal toxin development in beef sausages containing decolourized red blood cell fractions

Toxin production by Clostridium botulinum was studied in a model cured beef sausage containing decolourized dried bovine red blood cells (RBC), including intact RBC, acetone-treated RBC, enzyme-treated RBC, peroxide-treated RCB or plasma. Samples were formulated with beef shoulder, curing agents and spores of proteolytic strains of Clostridium botulinum. Vacuum packaged samples were heated to 72°C, stored at 28°C, and tested weekly. Sausages contained iron levels proportional to the iron in the blood fraction. Residual nitrite levels varied between < 10–40 μg g^-1. Toxin was detected earlier in samples containing higher levels of iron except for acetone-treated RBC. Higher pH values were associated with shorter times to toxin detection. We conclude that the RBC decolourization method can significantly modulate Cl. botulinum growth and toxigenesis.     

Hemolysis of rabbit erythrocytes in the presence of copper ions

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.     

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H₂O₂) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H₂O₂ molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H₂O₂ at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.     

Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins

The mechanism of the activation of prostaglandin endoperoxide synthetase by hemeproteins was investigated using the enzyme purified from bovine seminal vesicle microsomes. At pH 8, the maximal enzyme activities with methemoglobin (2 microM), indoleamine 2,3-dioxygenase (2 microM), and metmyoglobin (2 microM) were 70%, 42%, and 15% of that with 1 microM hematin. Apomyoglobin and apohemoglobin inhibited the enzyme activities caused by hemoproteins as well as that caused by hematin. The inhibition was removed by the addition of excess hematin. The dissociation of heme from hemoproteins was demonstrated by trapping the free heme with human albumin or to a DE-52 column. The dissociation of heme from methemoglobin was facilitated by increasing concentrations of arachidonic acid. The amount of heme dissociated from hemoproteins (methemoglobin, metmyoglobin, and indoleamine 2,3-dioxygenase) in the presence of arachidonic acid correlated with their stimulatory effects on the prostaglandin endoperoxide synthetase activity. Horseradish peroxidase and beef liver catalase, the hemes of which were not dissociated in the presence of arachidonic acid, were ineffective in activating prostaglandin endoperoxide synthetase. Spectrophotometric titration of prostaglandin endoperoxide synthetase with hematin demonstrated that the enzyme bound hematin at the ratio of 1 mol/mol with an association constant of 0.6 x 10(8) M-1. From these results, we conclude that hemoproteins themselves are ineffective in activating prostaglandin endoperoxide synthetase and free hematin dissociated from the hemoproteins by the interaction of arachidonic acid is the activating factor for the enzyme.     

Invertebrate red blood cell carbonic anhydrase

This is the first report documenting the presence of carbonic anhydrase (CA) for any invertebrate red cells. CA activity was measured in plasma, hemolysates of blood cells, and in hemolymph of selected species of invertebrates. Annelid red blood cells (RBC) and sipunculid pink blood cells both possessed significant levels of CA activity. Molluscan RBC, on the other hand, lacked CA activity. The distribution appears to have fallen along phylogenetic lines, with CA being present only in blood cells of the two more closely related groups. However, the presence of extracellular CA was confirmed in oyster hemolymph. Oyster hemolymph CA showed a similar affinity (Ki) for the sulfonamide inhibitors acetazolamide and ethoxzolamide, as did the vertebrate RBC CA II isozyme, supporting the idea that this isozyme could be the ancestral form of the enzyme.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.     

Lipid peroxidation and antioxidant systems in the blood of young rats subjected to chronic fluoride toxicity

Wistar albino rats were exposed to 30 or 100 ppm fluoride in drinking water during their fetal, weanling and post-weaning stages of life up to puberty. Extent of lipid peroxidation and response of the antioxidant systems in red blood cells and plasma to prolonged fluoride exposure were assessed in these rats in comparison to the control rats fed with permissible level (0.5 ppm) of fluoride. Rats treated with 100 ppm fluoride showed enhanced lipid peroxidation as evidenced by elevated malondialdehyde (MDA) levels in red blood cells but, 30 ppm fluoride did not cause any appreciable change in RBC MDA level. 30 ppm fluoride-intake resulted in increased levels of total and reduced glutathione in red blood cells and ascorbic acid in plasma while 100 ppm fluoride resulted in decreases in these levels. The activity of RBC glutathione peroxidase was elevated in both the fluoride-treated groups, more pronounced increase was seen with 100 ppm. Reduced to total glutathione ratio in RBC and uric acid levels in plasma decreased in both the groups. RBC superoxide dismutase activity decreased significantly on high-fluoride treatment. These results suggest that long-term high-fluoride intake at the early developing stages of life enhances oxidative stress in the blood, thereby disturbing the antioxidant defense of rats. Increased oxidative stress could be one of the mediating factors in the pathogenesis of toxic manifestations of fluoride.     

HDP-a novel heme detoxification protein from the malaria parasite

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous "Outbound-Inbound" trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target.     

Spectrophotometric method for determination of tocopherol in red blood cells

A relatively rapid procedure is described for the spectrophotometric determination of total tocopherol in red blood cells (RBC) based on a modification of the original Emmerie-Engel reaction. The critical feature in this method is the presence of a large amount of an added antioxidant, pyrogallol or ascorbic acid, during the saponification and extraction stages and the use of thin-layer chromatography for tocopherol purification. The total tocopherol levels of plasma and erythrocytes were determined for a number of human subjects, for patients with abetalipoproteinemia, and for rats. It was found that these levels had a wide range in normal human subjects but that the ratio of RBC to plasma tocopherol was relatively constant and equal to 0.18, uncorrected, and 0.21 when both RBC and plasma values were corrected to 100% recovery. The RBC-to-plasma ratio for rats was 0.39. The accuracy of this ratio determined by the spectrophotometric procedure was verified by measuring the distribution of [(14)C]tocopherol in RBC and plasma when radioactive vitamin E was introduced into the blood by both in vitro and in vivo techniques. The addition of radioactive tocopherol to RBC or plasma at the initial stage of the analysis permits an accurate determination of the total tocopherol in RBC or plasma by calculations based on the recovery of the added isotope. This procedure for erythrocyte tocopherol analysis is compared with a gas-liquid chromatographic method in current use.     

High plasma buffering and the absence of a red blood cell beta-NHE response in brown bullhead (Ameiurus nebulosus)

The high non-bicarbonate buffer capacity of brown bullhead (Ameiurus nebulosus) plasma was postulated to function as an alternative mechanism for the protection of red blood cell (RBC) intracellular pH (pHi) in the absence or attenuation of a RBC adrenergic response. The requirement for protecting RBC pHi arises from the presence of a Root effect haemoglobin in bullhead. In support of this hypothesis, bullhead RBCs incubated in vitro with isoproterenol (10(-8)-10(-5) mol l(-1)) or forskolin (10(-4) mol l(-1)) exhibited significant cyclic AMP accumulation, but failed to exhibit cell swelling or significant Na(+) or Cl(-) accumulation; plasma pH (pHe) was also unaffected. Similarly, no significant effect on RBC water content, Na(+) or Cl(-) concentration, or pHe was detected in bullhead blood incubated with 8-bromo cyclic AMP (10(-4)-10(-2) mol l(-1)) in vitro. These results suggest that while bullhead RBCs possess a beta-adrenoreceptor linked to cyclic AMP formation, stimulation of this adrenergic receptor does not result in measurable activation of a Na(+)/H(+) exchanger.