Studies on the Formation of Heinz Bodies. I. Methemoglobin Production and Oxyhemoglobin Destruction

A study of the changes in hemoglobin of erythrocytes incubated with varioustest substances has been reported. The concentrations of oxyhemoglobin andmethemoglobin in these erythrocytes were measured and the term "intact"hemoglobin was introduced, in order to distinguish these pigments from laterdegradation products of hemoglobin. Certain agents which are known to causeHeinz body formation and erythrocyte destruction in vivo have been shown tocause methemoglobin formation and "intact" hemoglobin destruction in vitro,and some of these agents were also shown to produce these changes in vivo.The possible significance of these findings in relation to the role of methemoglobin formation in Heinz body production has been discussed.

Submitted on May 26, 1960 Accepted on September 13, 1960     

Studies on Schmauch Bodies. I. The Incidence in Normal Cats (Felis Domestica) and the Morphologic Relationship to Heinz Bodies

Inclusions in the erythrocytes of normal cats, called Schmauch bodies, havebeen demonstrated by means of phase contrast microscopy and supravitalstaining. A very wide range of their incidence, but no correlation with theage of animals has been noted. By their identical staining reactions andcommon morphologic characteristics Schmauch and Heinz bodies are quitesimilar, although they might be produced by quite different mechanisms.

Submitted on July 8, 1964 Accepted on October 26, 1964     

The Attachment of Heinz Bodies to the Red Cell Membrane

S ummary . The binding between Heinz bodies or heat-precipitated haemoglobin and the red cell membrane was investigated. Ghosts containing Heinz bodies prepared from red cells containing Hb Christchurch and ghosts bound to heat-precipitated Hb A were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Using this technique, which separated the membrane and Heinz body proteins according to size, no protein bands corresponding to derivatives of haemoglobin covalently bound to a ghost protein were detected. Binding between red cell ghosts and heat-precipitated Hb A was also studied by density gradient ultracentrifugation, which separated ghosts bound to haemoglobin from pure ghosts and pure heat precipitate. Sulphydryl reducing and blocking agents were found to have no effect on the attachment of the insoluble haemoglobin to the ghosts, and reduction of electrostatic interactions in 2 m -NaCl was also ineffective.
It is concluded that covalent bonding, and in particular disulphide bonding, is not involved in the attachment of Heinz bodics to the red cell membrane. Hydrophobic bonding is suggested as the likely alternative.     

Heinz Body Anemia--An Ultrastructural Study. I. Heinz Body Formation

The ultrastructural changes in red blood cells exposed to phenylhydrazine,either in vivo or in vitro, are described. There is an age-dependent gradientof red cell sensitivity to this drug which includes the more mature reticulocytes as well as the population of circulating erythrocytes. Oxidative denaturation of hemoglobin and the formation of Heinz bodies, which constitute themajor drug-induced lesion, are accompanied by a regular sequence of structural changes commencing in the central cytoplasm of erythrocytes and thedrug-sensitive reticulocytes. These early changes often appear in close associaion with clusters of mitochondria. The initial morphologic lesion has anapparently crystalline structure and the significance of this stage is discussed.Heinz bodies grow by coalescence and condensation and finally come tolie just beneath the cell surface. Here they result in considerable distortionof cell shape and deformation of the plasma membrane. Thus, phenylhydrazine administration produces in red blood cells extensive ultrastructural alterations both in hemoglobin and in the cell membrane which may have considerable bearing on the fate of these cells in the circulation.

Submitted on September 29, 1964 Accepted on October 9, 1964     

Effect of Heinz Bodies on Red Cell Deformability

Decreased deformability of acetylphenylhydrazine-treated erythrocytes,measured by in vitro filtration experiments, was found to parallel Heinzbody formation. The decreased deformability seemed unrelated to methemoglobin formation, ATP depletion,red cell size, or membrane effect.Hemolysis of Heinz body erythrocytesoccurred during filtration of severelydamaged cells, in an all-or-nonefashion. In mixtures, separation of untreated cells from Heinz body-containing erythrocytes could be accomplished by filtration. There may be arelationship of these filtration properties to intravascular, in vivo destruction of severely damaged cells inHeinz body anemias.

Submitted on August 30, 1971 Revised on November 8, 1971 Accepted on November 9, 1971     

Phenyldiimide, Hemoglobin, and Heinz Bodies

Phenylhydrazine is oxidized stoichiometrically by two equivalents of ferricyanide to produce phenyldiimide and ferrocyanide. This reaction permits accurate determination of the absorption maxima and molar absorption coefficients of phenyldiimide in aqueous solution. These values are identical or extremely similar to those of phenyldiimide produced by decarboxylation of phenylazoformic acid. Knowledge of the stoichiometry of the reaction confirms an earlier suggestion that phenyldiimide is the product of ferricyanide-oxidized phenylhydrazine that reacts with heme proteins. Homologous structures postulated for phenyldiimideferrihemoglobin and nitrosobenzeneferrohemoglobin suggest a common factor in the production of Heinz bodies after exposure of erythrocytes to phenylhydrazine and to arylamines.     

Observations on Heinz Bodies in Normal and Splenectomized Rabbits

The disappearance from the circulation of Heinz bodies, produced byincubation of blood with phenylhydrazine, was studied in normal andsplenectomized rabbits. In the intact animal, red cells damaged by thisprocedure are removed largely by the spleen. In both intact and splenectomized rabbits, the Heinz bodies are withdrawn from the circulation byremoval in toto of the red cells containing them.

Submitted on January 9, 1959 Accepted on June 1, 1959     

Active Extrusion of Heinz Bodies in Drug-Induced Haemolytic Anaemia

S ummary . A patient with repeated episodes of sulphonamide-induced haemolytic anaemia is reported. The haemolytic episodes were associated with a high percentage of Heinz bodies which had the unique feature of being actively extruded from the erythrocytes. Detailed morphological and biochemical studies are outlined, but the basis for the tendency to form Heinz bodies remains unclear. It is suggested that the phenomenon of active extrusion may provide an alternative route, independent of the reticuloendothelial system, for the removal of Heinz bodies. The close resemblance between the active expulsion of the Heinz bodies and the physiological denucleation of the maturing erythroblast is noted and a similar mechanism postulated.     

On the Nature of Rheumatoid Rice Bodies

The nature of rice bodies was studied, utilizing histochemistry, immunofluorescence, and scanning and transmission electron microscopy. Rice bodies were found to consist primarily of fibrous material, most of which was fibrin with small amounts of collagen. Channels containing a variety of viable cells permeated the rice bodies. Blood vessels occurred in a few rice bodies indicating a former connection with the synovial membrane. Nonvascularized rice bodies might represent a further degeneration of the vascular type. Rice bodies seem to be a nonspecific response to inflammation.     

Methemoglobinemia, Heinz Bodies, and Acute Massive Intravascular Hemolysis in Lysol Poisoning

Two patients with poisoning by concentrated lysol are reported. One developed methemoglobinemia, a markeddecrease in red cell glutathione level, andlarge solitary Heinz bodies associatedwith massive intravascular hemolysis 3days later. The other patient, who hadabsorbed less lysol, developed methemoglobinemia and multiple small Heinzbodies that subsequently disappearedwithout frank hemolysis. No preexistingred cell defect was demonstrated in either patient, and in vitro experimentsshowed that these effects were due to adirect oxidant action of lysol and hydroquinone, a metabolite of phenol on thered cell.

Submitted on December 14, 1970 Revised on April 29, 1971 Accepted on July 16, 1971     

Unstable Hemoglobins: The Role of Heme Loss in Heinz Body Formation

Mutant, unstable hemoglobins precipitate as Heinz bodies in circulating red blood cells resulting in their premature hemolysis. We stress that generally these hemoglobins contain amino acid substitutions in the beta-chain of globin near the heme pocket, and demonstrate that heme binding suffers thereby. Four genetically unstable hemoglobins lost roughly half their heme content while precipitating into Heinz bodies. Conversely, repletion of hemes in vitro corrected the characteristically aberrant electrophoretic mobilities of these hemoglobins and concomitantly prevented their excessive denaturation into Heinz bodies. From the finding that heme-containing alpha-chains accumulate in solution during Heinz body formation, we propose that heme loss occurs predominantly from mutant beta-chains, which then precipitate. This mechanism of Heinz body formation is valid in most, but not all, the unstable hemoglobinopathies.     

Heinz Body Anemia: An Ultrastructural Study. II. Red Cell Sequestration and Destruction

This study reports electron microscope observations on the process of redcell sequestration and destruction in the spleen and liver of the phenylhydrazine-treated rabbit. Damaged red cells are recognized by virtue oftheir Heinz bodies, a morphologic manifestation of the oxidative injury whichthey have sustained. Sequestration, in the spleen, involves the selective accumulation of damaged cells within the vascular spaces of the Billroth cords.Erythrophagocytosis and the intracellular digestion of red cells followssequestration. More severely injured cells may undergo intravascular hemolysiswithin the splenic red pulp. In the liver, however, no evidence for the intravascular sequestration of injured red cells is observed. Damaged cells areremoved directly from the sinusoidal blood by erythrophagocytosis. Theselectivity of spleen and liver for red cells subjected to different degrees ofinjury is discussed in terms of the observed differences in the vasculararchitecture of the two organs.

Submitted on November 15, 1964 Accepted on November 22, 1964     

The Nature and Significance of Platelet Products in Human Plasma

Fresh plasma freed of intact platelets can be shown to contain minute particulate material (platelet-dust) which can be separated by ultracentrifugation. Much of this material is rich in phospholipid and shows coagulant properties resembling those of Platelet Factor 3. The supernatant plasma, freed of these particles, shows a corresponding deficiency in Platelet Factor 3 activity.
Electron microscopic studies suggest that the lipid-rich particles may originate from the osmophilic granules of platelets and that they are extruded on storage of platelets, even in the absence of coagulation. It is suggested that the liberation of this material is the basis of the process of 'activation' of platelets known to occur with storage.
When clotting occurs, coagulant particulate material is released from platelets, considerably in excess of what is required for thrombin generation and it can be detected in serum. Its presence accounts for the platelet-like activity of serum.
The material can be distinguished from intact platelets, red cell stroma and chylomicra. It was observed that chylomicra, freed of platelet-dust, were inactive in a thrombin generation test, which thus appeared responsive only to platelet type lipid. Both chylomicra and platelet-dust were effective in a Stypven time test. Attention is drawn to possible errors of interpretation which may arise from the use of the latter test as an index of Platelet Factor 3 activity.
Preliminary immunological investigation of platelets revealed the presence of fibrinogen and at least three other constitutents which appear to be primarily platelet components.     

Studies of Abnormal Leukocyte Bodies in the Mink

The occurrence of abnormal bodies in the cytoplasm of neutrophils, eosinophils, monocytes and lymphocytes of Aleutian (aa) mink is reported. Similarities of these bodies to the leukocyte abnormalities of Chediák-Higashi syndrome of children are pointed out.

Submitted on January 3, 1963 Accepted on April 23, 1963     

Experimental Studies on Erythropoietin. II. The Relationship between Juxtaglomerular Cells and Erythropoietin

The relationship between erythropoietin production and the juxtaglomerular(JG) cells of the kidney were studied using Harada’s method for staining andHartroft’s method for estimating JG granularity.

In stimulated erythropoiesis (after bleeding and phenylhydrazine injection),the JG cells also showed hypergranularity. In depressed erythropoiesis, onthe contrary, the JG cells showed hypogranularity. Furthermore, from theobservation of the chronological change of the JG cell, granularity tended tochange in parallel with erythropoietin production. This study gave an indirect evidence of the secretion of erythropoietin by juxtaglomerular cells ofthe kidney.

Submitted on October 17, 1961 Accepted on March 20, 1962