Oxidation and erythrocyte senescence.

Direct macrophage recognition of an externalized phosphatidylserine signal on senescent erythrocytes is a process of erythrophagocytic clearance that is in line with the general clearance process of all other circulating cells that become apoptotic. Advances in deciphering this process suggest that oxidation of the erythrocyte's hemoglobin, the salient target of the free radicals encountered in the circulatory environment, may drive subsequent steps. The progressive accumulation of oxidized hemoglobin covalently bound to the membrane skeleton not only disrupts membrane organization but also threatens eventual phospholipid oxidation via a calcium-promoted quasi-lipoxygenase activity. The emergence on the cell surface of a threshold concentration of oxidized phospholipids, principally phosphatidylserine, signals recognition by the CD36 macrophage receptor.

Exclusion of the stomatin, alpha-adducin and beta-adducin loci in a large kindred with dehydrated hereditary stomatocytosis.

Defects in stomatin, alpha-adducin, and beta-adducin have been implicated in erythrocyte disorders of cation permeability. We performed linkage analysis of the genetic loci for these proteins in a large kindred with xerocytosis (dehydrated hereditary stomatocytosis). Using polymerase chain reaction-based genotyping techniques, all three loci are excluded as disease gene candidates.

Porphyrin loading of lipofuscin granules in inflamed striated muscle.

To further the understanding of oxidative effects on inflammation injury to muscle fiber structure, fluorescent imaging analysis of human striated muscle tissues from a variety of inflammatory or postinflammatory etiologies was undertaken in a search for accumulated coproporphyrin, a red autofluorescent byproduct of heme biosynthesis that would theoretically be formed under oxidative insult. Using a differential excitation method of in situ analysis, porphyrin autofluorescence was detected in intact fibers within the context of the yellow autofluorescent subsarcolemmal lipofuscin granules. Relative measurements of porphyrin concentration in the granules from different patients indicated that the acute/subacute inflammatory specimens grouped significantly higher than the more chronic inflammatory and nonpathological specimens. Myoglobin was also found to be associated with the granules. Myoglobin heme iron could potentially serve as a Fenton reagent for the intracellular generation of hydroxyl radicals, which are responsible for the oxidation of the porphyrinogens. High-performance liquid chromatography analysis of extracted dense particles revealed coproporphyrin as the sole porphyrin present. The observation of coproporphyrin within lipofuscin granules, previously unreported, suggests that lipofuscin accumulation in striated muscle may begin under conditions of acute oxidative stress, as marked by the oxidation of extramitochondrial porphyrinogens that are immediately incorporated into the granules.

Membrane phospholipid asymmetry in human thalassemia.

Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe beta-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the alpha- or beta-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of beta-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound alpha-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.

Surface area and volume changes during maturation of reticulocytes in the circulation of the baboon.

Changes in the surface area and volume of reticulocytes were measured in vivo during late stage maturation. Baboons were treated with erythropoietin to produce mild reticulocytosis. Reticulocyte-rich cohorts of cells were obtained from whole blood by density gradient centrifugation. The cohorts were labeled with biotin, reinfused into the animal, and recovered from whole blood samples by panning on avidin supports. Changes in the surface area, volume, and membrane deformability were measured using micropipettes during the 2 to 6 weeks subsequent to reinfusion. For the entire cohort, the membrane area decreased by 10% to 15% and the cell volume decreased by approximately 8.5%, mostly within 24 hours after reinfusion. Estimates of the cellular dimensions of the reticulocyte subpopulation within this cohort indicated larger reductions in the mean cell area (12% to 30%) and mean cell volume (approximately 15%) of the reticulocytes themselves. Two weeks after reinfusion, the distribution of cell size for the cohort was indistinguishable from that of whole blood. There was evidence of slightly elevated membrane shear rigidity in some reticulocytes before reinfusion, but this slight increase disappeared within 24 hours after reinfusion. These are the first direct measurements of changes in the membrane physical properties of an identifiable cohort of reticulocytes as they mature in vivo.

The unusual pathobiology of hemoglobin constant spring red blood cells.

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other alpha-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV "normalizing" effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional alpha-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and alpha-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional alpha-thalassemic variants and found that in addition to oxidized beta-globin chains, oxidized alpha cs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized alpha cs- and beta-globin chains. The membrane alterations induced by alpha cs chains are more akin to those induced by beta A-globin chains than those induced by the alpha A-globin chains that accumulate in the beta-thalassemias. Thus, each globin chain, alpha cs, alpha A, beta A, appears to produce its own form of membrane perturbation.

Beta1 integrin-mediated adhesion between renal tubular cells after anoxic injury.

beta 1 integrin-mediated adhesion between renal tubular cells after anoxic injury. This study examined the effect of sublethal injury, induced by ATP depletion (5 mM cyanide in the absence of dextrose), on the distribution and function of beta 1 integrins in primary cultures of mouse proximal tubular (MPT) cells. It was shown in this study that sublethal injury results in loss of focal contacts present in uninjured MPT cells, and that the beta 1 integrin molecule becomes redistributed to the apical membrane domain of sublethally injured cells. Polystyrene beads coated with Arg-Gly-Asp (RGD)-containing peptide adhere to the surface of sublethally injured MPT cells but not to control, dextrose-treated cells, indicating that the beta 1 integrins present on the apical surface of the cell remain functional. The presence of an excess of free RGD-containing peptide reduces binding of RGD-coated beads to sublethally injured MPT cells by approximately 50%. It was also demonstrated that adherence of MPT cells in suspension to cyanide-treated monolayers is increased more than 300% above adhesion to control, uninjured monolayers. This abnormal cell-cell adhesion is ameliorated by the presence of an excess of RGD-containing peptide and is reversed if cyanide-treated cells are allowed to recover for 1 h. It was concluded that the beta 1 integrin becomes expressed on the apical surface of MPT cells after sublethal injury. These apically expressed integrins remain functional and mediate aberrant adhesion between MPT cells.

Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/- 0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role.

Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency.

Hereditary spherocytosis (HS) is a common hemolytic anemia of variable clinical expression. Pathogenesis of HS has been associated with defects of several red cell membrane proteins including erythroid band 3. We have studied erythrocyte membrane proteins in 166 families with autosomal dominant HS. We have detected relative deficiency of band 3 in 38 kindred (23%). Band 3 deficiency was invariably associated with mild autosomal dominant spherocytosis and with the presence of pincered red cells in the peripheral blood smears of unsplenectomized patients. We hypothesized that this phenotype is caused by band 3 gene defects. Therefore, we screened band 3 DNA from these 38 kindred for single strand conformational polymorphisms (SSCP). In addition to five mutations detected previously by SSCP screening of cDNA, we detected 13 new band 3 gene mutations in 14 kindred coinherited with HS. These novel mutations consisted of two distinct subsets. The first subset included seven nonsense and frameshift mutations that were all associated with the absence of the mutant mRNA allele from reticulocyte RNA, implicating decreased production and/or stability of mutant mRNA as the cause of decreased band 3 synthesis. The second group included five substitutions of highly conserved amino acids and one in-frame deletion. These six mutations were associated with the presence of comparable levels of normal and mutant band 3 mRNA. We suggest that these mutations interfere with band 3 biosynthesis leading thus to the decreased accumulation of the mutant band 3 allele in the plasma membrane.

Abnormal assembly of membrane proteins in erythroid progenitors of patients with beta-thalassemia major.

The life threatening anemia in beta-thalassemia major (Cooley's anemia) is characterized by profound intramedullary lysis, the cause of which is incompletely understood. Using marrow obtained from beta thalassemia major patients undergoing allogeneic bone marrow transplantation in Pesaro Italy, it became possible to directly study the mechanism of the intramedullary hemolysis. Based on our previous studies, we hypothesized that the unmatched alpha globin chains would interfere with normal assembly of erythroid precursor membrane proteins. Patient and control erythroid precursors were reacted with monospecific polyclonal rabbit antibodies directed against spectrin, band 3, and band 4.1 and with a monoclonal anti-alpha globin chain antibody. Using laser confocal fluorescence microscopy, normal erythroid precursors show no alpha globin chain accumulation and exhibited uniformly smooth rim fluorescence of the three membrane proteins. In some thalassemic precursors, spectrin appeared to interact with large alpha globin accumulations, and in many of these cells the spectin appeared clumped and discontinuous. Band 4.1 interacted strongly with accumulations of alpha globin in thalassemic precursors to produce bizarrely clumped zones of abnormal band 4.1 distribution. Band 3 was incorporated smoothly into thalassemic erythroblast membranes. However, the proerythroblasts and basophilic erythroblasts were significantly deficient in band 3. Thus, accumulations of alpha globin in beta-thalassemia major colocalized with and disrupt band 4.1 and spectrin assembly into the membrane. The cause of deficient band 3 incorporation into thalassemic proerythroblast membranes remains unknown. These profound membrane alterations would likely contribute to the intramedullary lysis seen in Cooley's anemia.

Leukemia with megakaryocytic differentiation following essential thrombocythemia and myelofibrosis. Case report and review of the literature.

Leukemias of megakaryocytic lineage are rare and heterogeneous clinical entities. The nomenclature published in the literature is confusing and perhaps inappropriate to designate these primary myeloproliferative disorders. We describe a patient with essential thrombocythemia who evolved through myelofibrosis and myeloid metaplasia to a final picture of leukemia with megakaryocytic differentiation in the peripheral blood. This case illustrates different aspects of a chronic myeloproliferative disorder where myelofibrosis and myeloid metaplasia are frequent but secondary events. We have reviewed the literature focusing on the role of clonal megakaryocytic proliferation in myelofibrosis and on the clinical characterization of leukemia with megakaryocytic phenotype. We also present our interpretation of the literature which indicates that a formal review of the nomenclature is urgently needed.

Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes.

The irreducible complexation of hemoglobin with spectrin is a natural phenomenon of red blood cell aging, positively correlating with increasing cell density and decreasing cell deformability. The current study begins to address the role of these complexes in the disruption of membrane skeletal physiology and structure. The effect of bound hemoglobin on spectrin dimer self-association was investigated in vitro. The extent of conversion of isolated spectrin dimers to tetramers was evaluated as a function of peroxide-induced globin complexation before the conversion incubations. The incremental accumulation of tetramer was observed to decrease with increasing peroxide concentration used in the globin complexation step. The role of oxidized heme in this process was made apparent by the inability of carboxyhemoglobin to inhibit tetramer accumulation. A Western blot analysis of naturally formed globin-spectrin conjugates demonstrated irreducible complexes of globin with both bands 1 and 2. The complexes are tentatively designated "h1" and "h2". This analysis also demonstrated that h1 is completely extractable from cell ghosts, whereas h2 is only 50% extractable. These findings are incorporated into a hypothesis linking globin-spectrin complexation and the consequent inhibition of spectrin dimer self-association to the clustered band 3 senescence antigen (Low et al, Science 227:531, 1985).

Continuous improvement, quality control, and cost containment in clinical laboratory testing. Effects of establishing and implementing guidelines for preoperative tests.

OBJECTIVE: To develop guidelines for laboratory tests ordered before admission for elective surgery. DESIGN: A seven-step continuous quality improvement process. SETTING: The Departments of Laboratory Medicine, Surgery, and Anesthesia of the University of Massachusetts Medical Center, a 384-bed, teaching, tertiary-care facility. PARTICIPANTS: Core group of the Department of Laboratory Medicine and the Laboratory Medical Advisory Committee. INTERVENTION: Guidelines were developed for laboratory tests ordered before elective surgery. They were divided into four major groups as well as by age and gender. After an intense educational effort, consent was obtained from the majority of surgeons, who agreed to delegate the ordering of tests to the nurses and anesthesiologists who examine patients before surgery. MAIN OUTCOME MEASURE: Charts chosen at random by the medical records department for the period prior to implementation of guidelines were reviewed and compared with records 1 and 2 years later. RESULTS: Reductions of 50% and 60% in the first and second years, respectively, in the overall number of tests ordered per patient were demonstrated. An improvement in the appropriateness of tests was also documented: 81% in the first year and 86% in the second year, compared with 65% appropriateness prior to implementation of guidelines. A 1-year savings of $66,981 and an overall 2-year savings of $75,995 were documented. CONCLUSIONS: We have described an approach that involves a sustained educational effort and collaboration of nurses and physicians and have presented specific guidelines for preoperative testing. A major decrease in the number of tests ordered, an increase in their appropriateness, and marked fiscal savings were documented.

A laboratory rotation for medical house officers. Bridging the gap.

In an attempt to improve physicians' laboratory practice behavior, the Department of Hospital Laboratories at the University of Massachusetts Medical Center developed a rotation for first year housestaff. Medical interns were chosen for this pilot program because they are the most frequent users of our laboratory facilities. Rotations provide an overview of the laboratory organization, quality control and assurance, appropriate use of laboratory testing, cost containment, and an introduction to different laboratory disciplines. As assessed by discussions during an interview following completion of the program, the participants have shown an increased understanding of how a modern hospital laboratory functions and of the complexity of services provided. The respect for the laboratory staff and confidence in test results issued have increased, and house officers are more likely to use laboratory services in a more cost-efficient manner.

Continuous improvement, quality control, and cost containment in clinical laboratory testing. Enhancement of physicians' laboratory-ordering practices.

In 1991, the University of Massachusetts Medical Center, in Worcester, developed a model for change by using a program of continuous quality improvement to enhance physicians' laboratory-ordering practices, particularly the test for bleeding times. We describe a model that was developed through a seven-step continuous quality improvement process, and we discuss our success in increasing the appropriateness of ordering the tests for bleeding times while significantly reducing the costs for patients and hospitals. The following factors contributed to the program's success: an advisory structure; presentations to the medical staff; focused feedback sessions; and most important, well-documented guidelines with institutional support for new behavior.

Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms for maximum activity.

The conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity under the conditions of high enzyme concentration (approximately 80 microM) required to measure initial rates for this poor substrate. The enzyme, as purified, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimulates amidation activity approximately 3000-fold at the optimum copper stoichiometry and the enzyme is then inhibited by excess Cu(II). No stimulation of amidation activity is observed upon the addition of the following divalent metal ions: Mn(II), Fe(II), Ni(II), Cd(II), and the oxovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-hydroxyhippuric acid to benzamide shows no dependence on copper, indicating that the copper dependence of the amidation reaction must be attributed to a copper dependence in peptide alpha-hydroxylation.

Influencing physician behavior with CQI: a case study.

Health care reform will require unprecedented levels of cooperation among physicians, health care administrators, and other providers in order to ensure high-quality, affordable care for all. At the University of Massachusetts Medical Center, CQI techniques helped engage physicians in an effort to substantially alter ordering patterns to cut costs and achieve quality goals.

Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley's anemia)

The profound and life-threatening anemia in patients with Cooley's anemia is ascribed primarily to intramedullary hemolysis (ineffective erythropoiesis), the cause of which is obscure. Based on prior morphologic data showing nuclear abnormalities, we hypothesized that accelerated apoptosis could occur in these erythroid precursors. The highly successful bone marrow (BM) transplantation program for patients with Cooley's anemia provided us with a unique opportunity to test this hypothesis. We obtained pretransplantation BM aspiration samples from patients undergoing BM transplantation in Pesaro, Italy and from their allogeneic donors. The erythroid precursors were isolated using ficoll sedimentation and then panning selecting fro CD45- cells. Cytospin and Giemsa staining showed that the separation provided greater than 90% erythroblasts. Five million of these erythroblasts were lysed and their DNA was isolated. There were obvious ladder patterns of DNA breakdown products in beta-thalassemia major samples, with less occurring in beta-thalassemia trait. Normal individuals showed only a slight smear of breakdown of DNA. These results indicate there is enhanced apoptosis in the erythroblasts in the BMs of Cooley's anemia patients. This finding might partially explain why most of these erythroblasts never survive to become mature erythrocytes.

Increased cisplatin tissue levels with prolonged arterial infusion in the rat.

Prolonged arterial infusions of cisplatin (DDP) have been effective in the treatment of regionally confined malignancies. It is unclear whether the route or schedule of DDP administration was responsible for the observed therapeutic benefit. To resolve this issue, tumor and normal tissue platinum (Pt) levels were determined in rats bearing hind-limb rat mammary tumors after intravenous (IV) and intra-arterial (IA) DDP infusions of constant dose and varying lengths. Infusions of DDP at 6 mg/kg were conducted IA over 30 minutes, and 3, 6, 24, and 48 hours and IV over 30 minutes and 48 hours. After infusion, Pt concentrations in solubilized tissue homogenates were measured by flameless atomic absorption spectroscopy. Maximum tumor Pt levels were seen after 48-hour IA infusion (29.3 micrograms Pt/mg tissue). IA infusions of 24 hours or less resulted in significantly lower Pt levels. Maximum tumor Pt concentration after IV administration was only 0.98 micrograms/mg tissue (48-hour infusion). Muscle Pt levels adjacent to the tumor were highest in the IA infused extremities, but at the 48-hour interval, were 53-fold less than tumor levels. Tumor and adjacent muscle Pt levels were not significantly different from each other after IV administration. This study provides pharmacologic evidence that lengthening the duration of IA DDP infusion increases tumor levels of Pt over that of IV or rapid IA administrations. The benefit of prolonged IA DDP infusions is dependent upon both route and schedule of drug administration.

High-performance tryptic mapping of recombinant bovine somatotropin.

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.