Hemoglobin Breakdown Research Articles

The magnetic resonance imaging of musculoskeletal hemorrhage.

Hematomas in the extremities can present clinically as a soft tissue mass. Hematomas can usually be distinguished from neoplasia on MR by the signal patterns of hemoglobin breakdown products, which are dependent on the chemical bonding and oxidation state of hemoglobin iron. Beginning with a discussion of relevant atomic electronic structure, this review will examine how oxyhemoglobin, deoxyhemoglobin, methemoglobin, and hemosiderin, the principal iron compounds occurring in the various stages of a hematoma, affect its appearance on MRI.

Some Women Pioneers In Haematology

Some Women Pioneers In Haematology

Modifications morphologiques de la métaphyse fémorale supérieure chez l'homme atteint de la maladie ostéoporotique

A study of cancellous bone in the femoral upper metaphysis was performed using a micro-camera. In osteoporotic disease, a qualitative disorganization of bone and vessels occurs. Bone lamellae break off or fracture. Lacunae fine down some bone plates. Connective tissue replaces part or the totality of the hard structure. Some vessels are functional, some are not. Some walls disappear and flux runs out of their lumen, producing hematomae. Quantitative bone loss results in cavities. Adipocyt cells fill in the gaps. Browish deposits settle on the lamellae. It is suggested that these are either settling stem pigments from the breakdown of hemoglobin or micro-thrombuses.

Intraocular mass simulating a retained foreign body

Purpose: To present an unusual case of a preretinal mass that simulated a retained metallic foreign body. Method: Case report. Results: A 30–year–old man presented with unilateral iridocyclitis and an ipsilateral preretinal mass with ultrasonographic and computed tomographic characteristics of a metallic foreign body. Histologic examination of the mass disclosed a central concentration of iron-containing hemoglobin breakdown products surrounded by a cocoon of fibrous tissue. Conclusion: Blood breakdown products surrounded by a fibrous capsule can present with the characteristics of an intraocular metallic foreign body.

Heme compounds in dinosaur trabecular bone.

Six independent lines of evidence point to the existence of heme-containing compounds and/or hemoglobin breakdown products in extracts of trabecular tissues of the large theropod dinosaur Tyrannosaurus rex. These include signatures from nuclear magnetic resonance and electron spin resonance that indicate the presence of a paramagnetic compound consistent with heme. In addition, UV/visible spectroscopy and high performance liquid chromatography data are consistent with the Soret absorbance characteristic of this molecule. Resonance Raman profiles are also consistent with a modified heme structure. Finally, when dinosaurian tissues were extracted for protein fragments and were used to immunize rats, the resulting antisera reacted positively with purified avian and mammalian hemoglobins. The most parsimonious explanation of this evidence is the presence of blood-derived hemoglobin compounds preserved in the dinosaurian tissues.

Identification of hemoglobin degradation products in Plasmodium falciparum

Malaria parasites break down human hemoglobin to its constituent amino acids by cysteine and aspartic proteinases. However, no one has previously been able to identify hemoglobin cleavage products in intact parasites. When isolated parasites were subjected to non-denaturing polyacrylamide gels electrophoresis, a unique protein band was found which contains heme and reacts with anti-human hemoglobin antibodies. This protein does not appear to represent oxidized or glycosylated hemoglobin, and is present in isolated parasites but not in the cytosol of infected or uninfected erythrocytes. When this band was eluted and subjected to SDS polyacrylamide gel electrophoresis, three bands were seen on Western blots. The proteins in these bands contain proteins with the N-terminal sequences of α- and β-globin chains but molecular masses of only 13.2–13.4 kDa. These data suggest that hemoglobin α- and β-chains are initially cleaved within the parasite phagolysosome to release peptides of 15–17 and 23–25 amino acids from the C-termini of α- and β-globin chains, respectively. Production of the hemoglobin breakdown products was inhibited by E-64, a cysteine proteinase inhibitor, suggesting the involvement of a cysteine proteinase in an early step of hemoglobin degradation.

Antimalarial chemotherapy based on proteinase inhibitors

During the erythrocytic phase of malaria, parasite proteinases are involved in the breakdown of haemoglobin, the re-invasion of erythrocytes by merozoites, and antigen maturation. Two factors are important in the design of compounds to inhibit them: (1) any parasite proteinase acting in the host's plasma must have a narrow substrate specificity if it is to escape the activities of the numerous proteinase inhibitors naturally present in the blood; and (2) inhibitors of parasite proteinase acting on intracellular targets must be able to enter host cells. By using serum-free medium supplemented with human, high-density lipoproteins in re-invasion experiments, a neutral cysteine proteinase, previously described as the merozoite proteinase for erythrocyte invasion (MPEI), was detected, both in free merozoites and the culture medium. Of various pseudopeptides tested as inhibitors of this enzyme, GlcA-Val-Leu-Gly- Lysψ [CH2-CO]C2H5 was the most efficient. This was used in the design of macromolecular inhibitors ...

Quinoline antimalarials: Mechanisms of action and resistance

The quinoline-containing antimalarial drugs, chloroquine, quinine and mefloquine, are a vital part of our chemotherapeutic armoury against malaria. These drugs are thought to act by interfering with the digestion of haemoglobin in the blood stages of the malaria life cycle. Chloroquine is a dibasic drug which diffuses down the pH gradient to accumulate about a 1000-fold in the acidic vacuole of the parasite. The high intravacuolar concentration of chloroquine is proposed to inhibit the polymerisation of haem. As a result, the haem which is released during haemoglobin breakdown builds up to poisonous levels, thereby killing the parasite with its own toxic waste. The more lipophilic quinolinemethanol drugs, mefloquine and quinine, are not concentrated so extensively in the food vacuole and probably have alternative sites of action. The technique of photoaffinity labelling has been used to identify a series of proteins which interact specifically with mefloquine. These studies have led us to speculate that the quinolinemethanols bind to high density lipoproteins in the serum and are delivered to the erythrocytes where they interact with an erythrocyte membrane protein, known as stomatin, and are then transferred to the intracellular parasite via a pathway used for the uptake of exogenous phospholipid. The final target(s) of quinine and melloquine action are not yet fully characterised, but may include parasite proteins with apparent molecular weights of 22 kDa and 36 kDa. As resistance to the quinoline antimalarials rises inexorably, there is an urgent need to understand the molecular basis for decreased drug sensitivity. A parasite-encoded homologue of P-glycoprotein has been implicated in the development of drug resistance, possibly by controlling the level of accumulation of the quinoline-containing drugs. As our molecular understanding of these processes increases, it should be possible to design novel antimalarial strategies which circumvent the problem of drug resistance.

Haemozoin: Identification and quantification

Haemozoin (malaria pigment) is a haem polymer resulting from the breakdown of haemoglobin by Plasmodium spp. This refractory substance has been the focus of many studies and of much debate, mainly because of its role in the pharmacological activity of certain antimalarials. Haemozoin is also important because its presence in tissues serves as an indicator of malaria infections, and may itself be a mediator of malaria pathogenesis. In this article, Amy Sullivan and Steven Meshnick review the structure and synthesis of haemozoin, and then focus on methods of haemozoin identification in tissue. This latter aspect has implications for the study of haemozoin both as an indicator of malaria infection and as a possible mediator of malaria pathogenesis.

EPR spectroscopic studies of haemoglobin breakdown in malarial parasite-infected erythrocytes

EPR spectra of an insoluble fraction from the rodent malarial parasite Plasmodium berghei, grown in mouse erythrocytes, have been measured. Signals due to high-spin iron(III) haem and two low-spin haem species were detected. Comparison with other derivatives of haemoglobin, and the use of a simple ligand-field model, identified the low-spin species as imidazole-hydroxy and bis-imidazole derivatives of haem. These species may represent intermediate stages in the breakdown of haemoglobin to the insoluble form, haemozoin. The utility of EPR spectroscopy for the study of the breakdown pathway is discussed.

Blepharoplasty: A Refined Technique Emphasizing Accuracy and Control

Although these techniques may seem demanding in controlling bleeding and avoiding use of local anesthetic, they provide distinct advantages: (1) Morbidity of tissue staining and subsequent ecchymosis is dramatically reduced. (2) Although edema still occurs, additional resolution time for breakdown of hemoglobin in the tissues is reduced, and the patient can resume normal activities with less camouflage makeup more rapidly. (3) Intraoperatively, lack of tissue staining and lack of distortion by local infiltration enable the surgeon to see anatomic structures, detail, and contour irregularities that are simply not visible or identifiable when infiltration has been performed, increasing both the accuracy and safety of the procedure. (4) Strict adherence to the principles of avoiding any dissection and of controlling bleeding deep to the plane of the orbital septum avoids the most dangerous potential complications of blepharoplasty while improving aesthetic results by avoiding overresection of soft-tissue components of the eyelids. (5) All of these techniques increase the accuracy, control, predictability, and safety of blepharoplasty.

Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites

The incidence of human malaria has increased during the past 20 years; 270 million people are now estimated to be infected with the parasite. An important contribution to this increase has been the appearance of malaria organisms resistant to quinoline-containing antimalarials such as chloroquine and quinine. These drugs accumulate in the acid food vacuoles of the intraerythrocytic-stage malaria parasite, although the mechanism of their specific toxicity in this organelle is uncertain. The primary function of the food vacuole is the proteolysis of ingested red cell haemoglobin to provide the growing parasite with essential amino acids. Haemoglobin breakdown in the food vacuole releases haem, which if soluble can damage biological membranes and inhibit a variety of enzymes. Rather than degrading or excreting the haem, the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble crystalline material called haemozoin or malaria pigment. These crystals form in the food vacuole of the parasite concomitant with haemoglobin degradation, where they remain until the infected red cell bursts. The structure of haemozoin comprises a polymer of haems linked between the central ferric ion of one haem and a carboxylate side-group oxygen of another. This structure does not form spontaneously from either free haem or haemoglobin under physiological conditions, and the biochemistry of its formation is unclear. Here we report the identification and characterization of a haem polymerase enzyme activity from extracts of Plasmodium falciparum trophozoites, and show that this enzyme is inhibited by quinoline-containing drugs such as chloroquine and quinine. This provides a possible explanation for the highly stage-specific antimalarial properties of these drugs.

脳代謝研究施設機能生化学部門最近の研究概説(I) -外傷性てんかんの成因に関する研究-

Post-traumatic epilepsy is characterized by epileptic seizures due to brain damage secondary to head injury, and it has been known since the days of Hippocrates. However, the pathogenesis of post-traumatic epilepsy in still unclear. Willmore et al (1978) first observed that injection of iron salts into the rat cortex induced a chronic epileptic focus (an experimental model of post-traumatic epilepsy). Head injury or hemorrhagic cortical infarction results in extravasation of blood and breakdown of red blood cells and hemoglobin. Biological iron is normally protein bound in hemoglobin and transferrin. Iron liberated from hemoglobin is thought to be associated with generation of active oxygen species. Moreover, hemoglobin itself may promote oxygen free radical generation. Oxygen free radicals, especially·OH is responsible for the induction of peroxidation of neuronal lipids, which causes damage to the neuronal membranes. On the other hand, ·OH accelerated the production of guanidino compounds in the brain, such as methylguanidine and guanidinoacetic acid. They are endogenous convulsants in the brain. These reactions may follow by excitatory and inhibitory neurotransmitter disorders, and may lead to development of epileptic discharges in the epileptogenic focus. Treatment with antioxidants such as epigallocatechin or a phosphate diester of vitamin E and C (EPC-K1), which are potent ·OH scavengers, may be useful for preventing development of the iron-induced epileptic focus.

Biochemical pathogenesis of post-traumatic epilepsy

Head trauma is often followed by epilepsy and may be related to the breakdown of red blood cells and hemoglobin within the CNS. Injection of hemoglobin or iron salts into the rat cortex is known to induce a chronic epileptic focus. We observed the formation of superoxide anion (O2) and hydroxyl radical (.OH) after ferric chloride injection into the rat cerebral cortex and suggest that these radicals, especially .OH, may be responsible for the initiation of lipid peroxidation in neuronal membranes and for the accelerated production of guanidine compounds in the brain, which may in turn lead to epileptogenicity. Then, we found that treatment with epigallocatechin (EGC) or a phosphate diester of vitamins E and C (EPC), which are potent .OH scavengers, significantly inhibited the formation of malondialdehyde and epileptic discharges in the iron-induced epileptic focus.

Involvement of the proteasome in various degradative processes in mammalian cells.

Eukaryotic cells contain a 700-kDa proteolytic complex (the "proteasome" or multicatalytic endopeptidase complex), whose role in intracellular protein breakdown is unclear. It has been suggested that the proteasome functions in the rapid degradation of oxidant-damaged proteins and in the ATP-dependent proteolytic pathway. To test these possibilities, oxidant-damaged hemoglobin and albumin were produced by treating hemoglobin and albumin with phenylhydrazine, with hydroxyl radicals, or with both hydroxyl and superoxide radicals. After oxidant damage, these proteins were degraded more rapidly in erythrocyte extracts and also by the purified proteasome. However, complete removal of proteasomes from these extracts by immunoprecipitation (or inhibitors of its proteolytic activity) did not reduce the breakdown of oxidant-damaged hemoglobin and decreased degradation of hydroxyl- and superoxide-treated proteins by only 30-40%. Thus, erythrocytes must contain another proteolytic system for degradation of oxidant-damaged proteins. In contrast, immunoprecipitation of proteasomes with polyclonal or monoclonal antibodies prevented the ATP/ubiquitin-dependent degradation of lysozyme and also blocked the ATP-stimulated degradation of ubiquitin-conjugated lysozyme in reticulocyte and skeletal muscle extracts. These data indicate a critical role of the proteasome in the degradation of ubiquitin-conjugated proteins and suggest that the proteasome is associated with or is a component of the larger ubiquitin-conjugate-degrading enzyme complex.

Polyamines inhibit the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates

Reticulocytes contain a soluble nonlysosomal proteolytic pathway that requires ATP and ubiquitin. Polyamines at physiological concentrations were found to inhibit rapidly the ATP-dependent proteolytic system in reticulocyte lysates; spermidine and putrescine inhibited this process by 26–72% and spermine by 71–96%. Spermine had little effect on the ATP-independent breakdown of oxidant-treated hemoglobin. By fractionating the ATP-dependent system, we show that polyamines inhibit the ATP-dependent degradation of ubiquitin-protein conjugates.

Free radical generating mechanisms in the colon: their role in the induction and promotion of colorectal cancer?

A hypothesis is presented to account for the dietary induction and promotion of colorectal cancer. The principal agents are the secondary bile acids, lithocholic and deoxycholic acids, the vitamin K group and ferrous iron complexes. These metabolites may interact to subvert the normal free radical generating mechanisms involved in mucosal defence. Diets high in fat and red meat and low in fibre support a Bacteroides-dominated colonic microflora, which both synthesis and utilises vitamin K2 isoprenalogues or menaquinones as enzyme co-factors. Iron(II) complexes such as haemin from the breakdown of dietary haemoglobin and myoglobin also serve as growth factors for these bacteria and provide a rich source of haem-iron for intestinal uptake. Biliary secretion is stimulated by dietary fat and bile acids are essential for the intestinal uptake of vitamin K and possibly of iron complexes such as haemin. In the mature colonocyte, vitamin K and haemin may initiate redox cycling reactions which liberate superoxide (O2-.). Bile acids can activate the membrane bound phospholipase to liberate arachidonate and diacylglycerol. This leads in turn to the production of more O2-. which can enter the microcirculation and acts as a potent chemoattractant for the neutrophils that line the lamina propria. The released diacylglycerol can activate protein kinase C in the neutrophil membrane to switch on the respiratory burst oxidase system generating yet more O2-. and may stimulate the proliferation of transformed stem cells by a similar protein kinase C mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite.

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.

Bilirubin and the induction of intracranial arterial spasm.

Although a number of substances which participate in the physiological control of vascular caliber have been proposed to play a causative role in cerebral vasospasm, none of these has been shown to induce the profound, sustained degree of constriction or the vasculopathy that characterize this disorder. The present study was undertaken to determine whether bilirubin, a hemoglobin breakdown product with detergent-like activity, accrued in incubated blood or in intracranial hematoma and whether topical application of bilirubin altered the caliber or morphology of cerebral arteries. As a model of blood residing in the subarachnoid space, sterile vials of cat blood and of human blood were incubated in vitro at 37 degrees C. The concentration of bilirubin in the supernatant of cat blood increased from a value of 0.27 +/- 0.3 mg% (mean +/- standard error of the mean) on Day 0 to a value of 9.57 +/- 2.4 mg% on Day 10; the respective values for human blood were 0.53 +/- 0.02 mg% and 13.4 +/- 5.4 mg%. Samples of intracranial hematoma that had been surgically removed from three patients between 4 and 11 days after hemorrhage yielded bilirubin levels from 2.1 to 15.2 mg%. Application of a bilirubin suspension of 5, 10, or 20 mg% in buffered Ringer's solution to cat basilar artery in vivo led to progressive and sustained constriction; at 4 hours the mean decrease in the width of the blood column was 34% +/- 2.1%. Ultrastructural analysis of these vessels showed widespread pathological changes similar to those associated with cerebral vasospasm. Application of a 10-mg% bilirubin suspension to the basilar artery of two baboons produced similar alterations. Minimal changes in diameter or morphology were observed in cat arteries bathed in buffer solution alone. These findings in animal models indicate that bilirubin may play a central role in the development of cerebral vasospasm.

Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin.

It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins.