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     

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.     

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 the Formation of Heinz Bodies. II. The Nature and Significance of Heinz Bodies

Simultaneous studies were made as to the degrees of Heinz body formation,of change in hemoglobin and of change in osmotic fragility in erythrocytes incubated with certain drugs which are known to produce Heinz bodies in vivo.Certain factors that modify the pattern of effect of drugs on normal adult erythrocytes were also studied.

A close relationship was found between the degrees of Heinz body production and "intact" hemoglobin destruction. Factors that caused variation in thedegree of "intact" hemoglobin destruction caused similar variation in thedegree of Heinz body production. Staining studies suggested that Heinzbodies are protein in nature and derived from hemoglobin. These observationswere taken to indicate that Heinz bodies probably result from the direct effectof certain drugs on intra-erythrocytic hemoglobin.

-naphthol and primaquine were found to cause marked increase in osmoticfragility relative to the degrees of Heinz body production and of "intact" hemoglobin destruction, whereas acetyl phenylhydrazine produced marked Heinzbody production and "intact" hemoglobin destruction with relatively littleincrease in osmotic fragility. These observations were regarded as evidencethat Heinz body production is not the sole determinant of instability of erythrocytes exposed to these various drugs.

Under no experimental conditions was Heinz body production or "intact"hemoglobin destruction found without methemoglobin formation. Evidencewas presented to indicate that Heinz body production could be inhibited bytrapping methemoglobin as methemoglobin cyanide or methemoglobin azide.The suggestion was advanced that methemoglobin is in fact an essential stagein the destruction of "intact" hemoglobin and the formation of Heinz bodies.

Submitted on October 6, 1960 Accepted on January 12, 1961     

Protection by Ascorbate against Acetylphenylhydrazine-Induced Heinz Body Formation in Glucose-6-Phosphate Dehydrogenase Deficient Erythrocytes

Ascorbate was found to inhibit oxidation of oxyhaemoglobin and Heinz body formation in glucose-6-phosphate dehydrogenase deficient red cells incubated with acetylphenylhydrazine. It is proposed that ascorbate can substitute for the glutathione which is depleted and act as a scavenger for drug free radicals generated during the reaction. Ascorbate was protective at concentrations only a little higher than that in normal blood, and the possibility that administration of ascorbate could protect GSH deficient cells against the action of oxidative drugs such as APH is considered. A simple method of quantitatively assessing Heinz body formation by measuring the turbidity of the lysed cells is described.     

The Role of Disulphide Bonds in Heinz Body Attachment to Membranes

The mechanism of binding of oxidant-induced Heinz bodies to red cell membrane was studied. Heinz bodies induced by acetylphenylhydrazine in intact cells or in the presence of membrane remained attached to membrane when separated by a sucrose gradient. Incubation with sulphydryl reagents failed to free intact Heinz bodies from or prevent attachment to membranes, although the amount of haem was reduced. Thus disulphide bonds do not appear to be a major mechanism of attachment of Heinz bodies to red cell membrane.     

Salicylazosulphapyridine-induced Heinz body anemia.

A 58-year-old patient suffered from severe malabsorption due to agammaglobulinemia. Treated empirically with salicylazosulfapyridine 2--6 g/day, the subjective and objective features of malabsorption regressed. About a year after this treatment she developed a generalized weakness without renewal of the diarrhea; the diagnosis of Heinz body hemolytic anemia was established. In our patient, hemolysis began many months after the commencement of treatment and no deficiency of G6PD or other erythrocyte enzyme or pathological hemoglobin were found.     

Proteoglycans and Hypertension I.A Biochemical and Ultrastructural Study of Aorta Glycosaminoglycans in Spontaneously Hypertensive Rats

The extracellular matrix of blood vessel walls contains elastin, collagen, and proteoglycans, all of which can affect vascular resistance and, hence, blood pressure by virtue of their biomechanical properties.In the present study, we have begun to explore the possibility that proteoglycans may play a role in the pathophysiology of hypertension by analyzing, qualitatively and quantitatively, the polysaccharide components of proteoglycans from aorta of two normotensive rat strains, Wistar Kyoto (WKY) and Wistar rats, and from spontaneously hypertensive (SH) rats of the Okamoto strain.The total concentration of aorta glycosaminoglycans in the SH rat was 33% higher than in the WKY rat, due to a 164% increase in chondroitin 4- and 6-sulfate.The content of dermatan sulfate (DS), hyaluronic acid (HA), and heparan sulfate (HS) was similar in the two strains.The 4-wk-old SH rat also had an increase in chondroitin sulfate (CS) compared to the 4-wk-old WKY rat, without any change in DS, HA, or HS.The Wistar rat had approximately the same concentration of CS and DS in the aorta as the WKY rat, but HS and HA were reduced by 62 and 37%, respectively.The galactosaminoglycans (CS and DS) were heterogeneous on cellulose acetate electrophoresis and exhibited a different pattern for each of the three strains.Undersulfated CS accounted for 15 % of the total CS in WKY aorta but was present in only trace amounts in the SH aorta; 2% of the CS from the Wistar aorta was undersulfated.In all three strains, DS was exclusively 4-sulfated, and the CS contained approximately equal amounts of 4- and 6-sulfated galactosamine residues.Ultrastructural studies demonstrated that the HS was localized in the subendothelial matrix and the pericellular region surrounding the medial smooth muscle cells.CS and DS were primarily associated with collagen in the media.In the SH rat aorta the subendothelial matrix was thicker, and there was a relative increase in the CS/DS in the smooth muscle cell pericellular matrix.We suggest that, if similar alterations in CS proteoglycans are present in the resistance vessels, these changes may contribute to the increased peripheral vascular resistance in the hypertensive animal.     

Ultrastructural Observations on Platelet Adhesion Reactions: I. Platelet-Fibrin Interaction

The morphology of platelet-fibrin interaction in retracting clots and followingexposure of platelets to preformed fibrin particles has been studied with theelectron microscope. The interaction involved close conformity of the structuresat a finite distance, usually 100 to 200 angstroms, and platelet engulfment offibrin. The morphology closely resembles other platelet adhesion reactions, andit is suggested that the interaction described here is the ultrastructural representation of platelet-fibrin adhesion.

Submitted on May 19, 1966 Accepted on October 4, 1966     

Body weight and psychological distress in NHANES I.

This study examined the relationship between body mass index, smoking status, and depressive symptoms reported on the Center for Epidemiologic Studies Depression (CES-D) scale in the First National Health and Nutrition Examination Survey (NHANES I). Among women, but not men, greater body mass index was weakly associated with elevated reports of depressive symptoms. This relationship remained significant after controlling for age, years of education, and smoking status. A history of smoking (current and ex-smoking) and body weight in the highest weight quintile (> or = 28.96 kg/m2) was marginally related to increased risk of depression (CES-D score > or = 16) among women only. These results indicate that relative body weight is weakly related to psychological distress among women but not men, and that cigarette smoking does not significantly modify this relationship.     

Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization

Objective. To examine the ultrastructure of human synovial mast cells in situ, to identify immunologic and nonimmunologic stimuli that activate these cells in vitro, and to quantify a number of preformed and de novo–synthesized mediators. Methods. We conducted an ultrastructural study of synovial mast cells in situ and performed immunoelectron microscopy localization of tryptase and chymase. Isolated synovial mast cells were analyzed biochemically, immunologically, and functionally in vitro and compared with cells from human lung, heart, and skin. Results. Ultrastructural study of synovial tissue revealed mast cells with homogeneously dense, scrolled, crystal, and mixed granules, and lipid bodies in the cytoplasm. A small percentage of mast cells showed evidence of degranulation. Immunoelectron microscopy demonstrated the subcellular localization of tryptase and chymase over granules of >90% of the mast cells, which were of the MC_TC subtype. Isolated synovial mast cells released histamine in response to immunologic (anti-IgE and anti-Fcε receptor I [anti-FcεRI]) and nonimmunologic (substance P, recombinant human stem cell factor, and 48/80) stimuli, but did not respond to recombinant human C5a in vitro. Synovial mast cells differed from those isolated from other human tissues, in a variety of immunologic and biochemical features. There was a linear correlation between the percentage of histamine secretion and tryptase release (r = 0.79, P < 0.001) induced by cross-linking of FcεRI. Cross-linking of IgE with anti-IgE on synovial mast cells induced de novo synthesis of prostaglandin D₂ (mean ± SEM 87.5 ± 4.9 ng/10⁶ cells) and of leukotriene C₄ (57.6 ± 17.8 ng/10⁶ cells). Conclusion. Mast cells ultrastructurally characterized in situ in synovial tissue were seen to differ from mast cells previously isolated from other human tissues. This raises the possibility that the local microenviroment influences their phenotype. Isolation of mast cells from human synovia can be useful for studying their role and their mediatrors in patients with arthritis.     

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     

Recovery of the heart after normothermic ischemia. Part I: Ultrastructural findings during postischemic reperfusion.

The influence of controlled ischemia on myocardial ultrastructure was investigated in isolated, metabolically supported canine hearts. Recovery of functionally normal tissue as indicated by the reversibility of morphological alterations was observed up to 60 minutes of anoxia. It was shown that prolonged reperfusion of the empty beating heart supports the recovery of normal cellular ultrastructure. Severe ischemic damage of mitochondria due to ischemia of 60 minutes was almost completely reversible after a reperfusion period of 50 minutes.