Inhibition of synthesis of fetal hemoglobin by an isoleucine analogue.

Reticulocytes from newborn infants with Rh isoimmune hemolytic disease actively incorporated radioactive amino acids in vitro into hemoglobins F and A. Approximately 50% of the reticulocytes appeared capable of synthesis of both of these hemoglobins within the same cell, as demonstrated by the selective elution technique of Betke and Kleihauer. An isoleucine analogue, L-O-methylthreonine, inhibited the incorporation of a variety of amino acids into hemoglobin F, without significantly affecting the synthesis of hemoglobin A. The inhibition was prevented upon concomitant addition of L-isoleucine to the medium. These observations suggest that an independent biosynthetic apparatus is present in the cell for the synthesis of each of these two hemoglobins. Because isoleucine is present only in the gamma chains of hemoglobin F, the inhibitory effect of the analogue on the synthesis of this hemoglobin must represent a selective effect on the production of gamma chains.

Hemoglobin Abraham Lincoln, beta32 (B14) leucine leads to proline. An unstable variant producing severe hemolytic disease.

An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t(1/2) using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[(14)C]leucine indicated a t(1/2) of 7.2 days. When stroma-free hemolysates were heated at 50 degrees C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37 degrees C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant beta-chain by precipitation with p-hydroxymercuribenzoate. Purification of the beta-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for beta32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at beta31 to impair heme stabilization.

Hemoglobin Evanston (alpha 14 Trp----Arg). An unstable alpha-chain variant expressed as alpha-thalassemia.

A new hematologic syndrome with phenotypic features of mild Hb H disease was identified in three children from two unrelated black American families. Erythrocytes from each of these children contained Hb H (beta 4) and Hb Barts (gamma 4), as well as a slowly migrating hemoglobin fraction that made up 7-10% of the total hemoglobin. The parents of the affected children all showed mild thalassemia-like changes, with one of the parents in each family also expressing the variant hemoglobin; in the latter individuals the mutant alpha-chains made up less than 2% of the total, and were present mainly or exclusively in combination with delta-chains in the form of a slowly migrating Hb A2. Purified Hb Evanston showed an increased oxygen affinity, but its Bohr effect, cooperativity, and 2,3-diphosphoglycerate effect were normal. The mutant hemoglobin appeared to have normal stability to heat and to isopropanol, and the stability of its alpha-chain in an extended time course synthesis study also appeared to be similar to that of alpha A. However, the results from short-term globin synthesis studies, and from mRNA translation in vitro, suggest that the two types of alpha-chains were synthesized at relatively equal rates, with a major fraction of the newly synthesized variant alpha-chains undergoing rapid catabolism. The hematologic data taken in combination with DNA hybridization and globin synthesis findings indicate that the proposita in each of these families has the genotype--, alpha A/--, alpha Ev. These observations suggest that two separate mechanisms are contributing to the alpha-thalassemia-like expression of Hb Evanston : the newly synthesized alpha EV-chains are unstable and are subject to early proteolytic destruction; and the mutant alpha-allele is linked to an alpha-globin gene deletion.

Hemoglobin Milledgeville (alpha 44 (CD2) Pro leads to Leu): a new variant with increased oxygen affinity.

Hemoglobin Milledgeville, a new hemoglobin structural variant, was identified in three members of a black American family. The oxygen affinity of blood and hemoglobin samples from the affected individuals was markedly increased (p50 O2 of whole blood 11-15 mmHg at 37 degrees C, pH 7.4), and the abnormality was associated with mild erythrocytosis. The variant hemoglobin did not separate from Hb A by electrophoresis or by chromatography or isoelectric focusing, and efforts to isolate an abnormal globin chain were also unsuccessful. The Hb A2 fraction as well as Hb A from erythrocytes of affected individuals exhibited increased oxygen affinity, indicating that the altered oxygen equilibrium was the result of a hemoglobin alpha chain abnormality. Fractionation of trypsin and chymotrypsin digests of isolated alpha chains demonstrated a single abnormal peptide representing a Pro leads to Leu substitution at alpha 44 (CD2). Properties of Hb Milledgeville include low cooperativity (n = 1.1-1.4), a normal alkaline Bohr effect (delta logp50/delta pH = -0.62), and normal interaction with 2,3-diphosphoglycerate. The alpha CD2 proline residue normally participates in the formation of the alpha 1 beta 2 subunit interface in the deoxy quaternary conformation, but not in oxyhemoglobin; the leucine substitution may produce destabilization of the deoxy conformation with a resulting shift in equilibrium toward the oxy conformation.

Hemoglobin F Lódz (G gamma I 44 Ser replaced by Arg). A newly identified variant from an American infant of Polish descent.

An electrophoretically slowly migrating hemoglobin variant was identified in a neonate of Polish parents from a cord blood hemoglobin survey. Structural analysis of the gamma chain of the mutant hemoglobin, with fractionation of the tryptic peptides by means of high-performance liquid chromatography, demonstrated a substitution of serine-44 by arginine. Glycine was identified at position 136 of the gamma chain and isoleucine at position 75. Serine residue 44 appears not to be a heme contact site, but as a result of the substitution in this mutant hemoglobin, the possibility exists for formation of a salt bridge between the arginine and a heme propionate group. The infant's hematologic findings suggested the possibility of mild hemolysis, but these changes may have been due to isoimmune disease.

Iron deficiency and sickle cell anemia.

In a patient with sickle cell anemia, iron deficiency was accompanied by hypochromic, microcytic RBCs, absence of bone marrow iron, and a low serum ferritin level. The mean corpuscular hemoglobin concentration (MCHC) was decreased (27.6 g/dL) and was associated with an extreme scarcity of sickled erythrocytes in blood smears. Iron therapy resulted in reticulocytosis and an increase in sickled erythrocytes. In vitro studies demonstrated a decrease in sickling of erythrocytes as a function of oxygen saturation of the blood when the patient was iron deficient. The whole blood oxygen dissociation curve showed a substantial decrease in oxygen pressure necessary to produce 50% saturation of hemoglobin at pH 7.4 and 37 degrees C (P50), indicating an increased oxygen affinity. These data suggest that a reduction of the MCHC induced by iron deficiency may ameliorate sickling.

Sickle cell anemia in two White American children: essential laboratory criteria for diagnosis.

A number of hematologic disorders share diagnostic and clinical features of sickle cell anemia but have significantly different genetic implications and prognosis. Because of these differences, the establishment of a precise diagnosis is essential for the child in whom any form of sickle cell disease is identified. To illustrate the requirements for a definitive laboratory diagnosis of sickle cell anemia, this report presents the approach to establishing this diagnosis in two white American patients. From a review of the literature, these patients appear to be the only white Americans with sickle cell anemia in whom the diagnosis has been unequivocally established.

Modelling whole blood oxygen equilibrium: comparison of nine different models fitted to normal human data.

The ability of nine different models, prominent in the literature, to meaningfully characterize the oxygen-hemoglobin equilibrium curve (OHEC) of normal individuals was examined. Previously reported data (N = 33), obtained using the DCA-1 (Radiometer, Copenhagen), and new data (N = 8), obtained using the Hemox-Analyzer (TCS, Southampton, PA), from blood samples of normal, non-smoking volunteers were used and these devices were found to give statistically similar results. The OHECs were digitized and fitted to the models using least-squares techniques developed in this laboratory. The "goodness-of-fit" was determined by the root-mean-squared (RMS) error, the number of parameters, and the parameter redundancy, i.e., correlation between the parameters. The best RMS error did not necessarily indicate the best model. Most literature models consist of ratios of similar-order polynomials. These showed considerable parameter redundancy which made the curve fitting difficult. The best fits gave RMS errors as low as 0.2% saturation. The Hill model gave a good characterization over the saturation range 20%-98% with RMS errors of about 0.6% saturation. On the other hand, good characterizations over the entire range were given by several other models. The relative advantages and disadvantages of each model have been compared as well as the difficulties in fitting several of the models. No single model is best under all circumstances. The best model depends upon the particular circumstances for which it is to be utilized.

Sickling syndromes in children.

The sickle cell syndromes comprise a highly diverse group of disorders which have in common the presence of sickle hemoglobin in the blood erythrocytes. The establishment of a precise diagnoffort involving both the patient and his family, but because of the important differences in prognosis and management of these various syndromes, an accurate diagnosis is of fundamental importance. Definitive therapy has not yet been developed for any of these disorders, but with the application of available forms of treatment these patients can be benefited substantially.

beta-Thalassemia arising as a new mutation in an American child.

A 6-year-old child of northern European ancestry was found to have microcytic, hypochromic anemia with an elevated level of hemoglobin A2 and an unbalanced pattern of globin chain synthesis characteristic of beta-thalassemia trait. Hematologic and globin synthesis studies of both parents yielded entirely normal results. Identification of the mother and father as the biological parents was established with a high order of reliability by determination of erythrocyte, serum, and HLA genetic markers. These findings suggest that the picture of beta-thalassemia observed in this child represents a new mutation.

Hemoglobin C-I-A.

Hemoglobin I (alpha 16 lys leads to glu) was identified in association with hemoglobins C and A in a black American woman who had no clinical or hematologic disease. Her erythrocytes contained four major hemoglobins, all of which were separable by DEAE-cellulose column chromatography. Nearly 50% of the total recovered alpha I-chains were present in combination with beta C, suggesting that there may have been preferential formation of the alpha I beta C hybrid hemoglobin.

Fever and neutropenia in children with neoplastic disease: an analysis of 158 episodes.

The development of fever in neutropenic cancer patients is frequently an indication of infection. The response of these patients to antibiotic treatment is often poor, and the recent literature continues to document a substantial mortality rate in spite of the prompt treatment of these patients with multiple-agent antibiotic therapy. Because of limited available information regarding fever and neutropenia in pediatric cancer patients, we undertook an analysis of a group of such patients from a pediatric oncology center. The incidence of probable and documented infections was 39.2% in a group of these patients, representing 158 febrile episodes. The most frequent form of infection was bacterial sepsis; pulmonary infections were the next most frequent, followed by infections of skin and soft tissues. In leukemia patients with culture-proven infections, gram-negative organisms were isolated in 61.1% of episodes while gram-positive organisms were isolated in 41.7%. Escherichia coli and Staphylococcus aureus were the organisms most frequently isolated from these patients. In solid-tumor patients with bacterial infections, gram-positive organisms were isolated in 78% of cases. Patients having the highest incidence of documented infections were those with leukemia who had active disease (induction or relapse), and severe neutropenia (less than 200 granulocytes/microliter). Antibiotic therapy with cephalothin, gentamicin, and carbenicillin (CGC), was effective in 41 of 45 (91.1%) episodes of documented infection in the total group of patients.