The xerocytosis of Hb SC disease

Patients with Hb SC disease were found to have microcytic and hyperchromic red cell indices despite mild reticulocytosis. Iron deficiency anemia was ruled out by the finding of normal serum ferritin levels. In order to determine whether the microcytosis was due to coexistent alpha-thalassemia, restriction endonuclease mapping was performed on genomic DNA extracted from peripheral blood leukocytes. Patients with Hb SC disease had microcytic indices despite the presence of a full complement of four alpha-genes (alpha alpha/alpha alpha), suggesting that the microcytosis may be due to cellular dehydration (or xerocytosis), since the mean corpuscular hemoglobin concentration in Hb SC disease patients was significantly higher than in controls. This possibility was investigated further by the determination of RBC cation content. RBC Na levels were similar in SC and normal red cells. Hb SC RBCs, however, had significantly reduced K levels. These findings show that RBC cation content, and thus cell water, is decreased in Hb SC disease. The decreased RBC K level in the presence of normal cellular Na concentration suggests selective K loss that is not due to inhibition of the Na K pump. Ouabain-insensitive K+ efflux was increased to four times normal in SC cells. Cell dehydration was confirmed by the demonstration of increased high-density RBCs on discontinuous Stractan density gradients and by osmotic gradient ektacytometry. Cellular dehydration and its sequelae were worse in CC erythrocytes and milder in AC cells than in Hb SC red cells. Taken together, these data indicate that in Hb SC disease the RBCs are severely dehydrated and typically microcytic and hyperchromic. Hb SC RBCs seem to be dehydrated due to selective K loss. These findings suggest a functional interrelationship between Hb SC, the red cell membrane, and cation regulation.     

Rheologic predictors of the severity of the painful sickle cell crisis

Deformable sickle erythrocytes have been reported by Mohandas and Evans to be more adherent to vascular endothelium than rigid irreversibly sickled cells (ISC). To define the clinical implications of this finding we have determined genetic, hematological, clinical, and rheological characteristics of sickle erythrocytes obtained from 65 patients with sickle cell anemia and fetal hemoglobin (Hb F) levels less than 15%. The alpha-globin gene number had a significant effect on the hematological parameters, the percentage of dense cells, ISC number, and HB A2 levels. The presence or absence of alpha thalassemia, however, had no effect on the frequency and severity of the sickle cell painful crisis (r = 0.06, P greater than .05). RBC deformability, determined by an ektacytometer, showed great heterogeneity among patients with three or four alpha-globin genes. Linear regression analyses of the data showed significant positive correlation of the frequency and severity of the painful crisis with RBC deformability (r = 0.49, P less than .001), and negative correlations with the percentage of dense cells (r = -0.37, P = .002), and the percentage of ISC (r = -0.46, P less than .001). We propose that the more deformable the sickle RBC are, the greater their adherence to vascular endothelium, and the more they cause vaso-occlusive crises, RBC deformability and the percentage of dense cells (or ISC) seem to have a predictive value of the frequency and severity of painful crises in sickle cell anemia.     

Blood cell values in healthy Hong Kong Chinese adults

Haemoglobin concentration and other blood cell indices were determined in a Coulter counter (Model S-Sr) in order to establish a reference range for healthy Hong Kong Chinese adults. The mean values for all the blood cell indices were significantly higher (P less than 0.0005) in males than in females, except for MCHC where there was no difference. In males, there was a statistically significant age-related decrease in RBC counts and Hb concentration, whereas MCV showed a statistically significant increase with age. The WBC counts also showed a statistically significant increase with age. In females, however, all the blood cell indices remained relatively unchanged up to 70 years of age, after which there was a drop in the RBC counts (P less than 0.01), PVC (P less than 0.05) and Hb concentration, the latter not reaching statistical significance. The MCH, however, increased after 70 years of age (P less than 0.05). The reference ranges derived for healthy Hong Kong Chinese adults are of a similar order of magnitude to those already published.     

Alpha Thalassaemia in Sicily: Haematological and Biosynthetic Studies

Eight Sicilian patients with Hb H disease and their families have been studied. The standard haematological tests and the alpha/beta chain synthesis ratios showed significantly different results in the patients with Hb H disease as compared with alpha thalassaemia carriers, except for Hb A2 values. There was no significant difference in the mean RBC, MCV, Hb A2, Hb A1 and Hb F of alpha thalassaemia carriers compared with normal controls. On the contrary significant difference was found between the mean alpha/beta chain synthesis ratio of alpha thalassaemia carriers and that of the normal controls; however, the extensive overlapping of alpha/beta values between these two conditions make this parameter insufficiently discriminant. No correlation was found between MCV, MCH, RBC and alpha/beta chain synthesis ratio in patients with alpha thalassaemia trait, suggesting that the ratio cannot be used to distinguish between carriers of a mild gene ('silent' carrier) and carriers of the more severe alpha thalassaemia gene. A possible genetic model for alpha thalassaemia in Sicily is presented.     

Molecular and cellular pathogenesis of hemoglobin SC disease.

Solution and cell studies were performed to ascertain why individuals with hemoglobin (Hb) SC have disease whereas those with Hb AS do not. The polymerization of deoxygenated mixtures containing sickle cell Hb (Hb S; alpha 2 beta 2(6)Glu leads to Val) and Hb C (alpha 2 beta 2(6)Glu leads to Lys) was investigated by measurements of delay times and solubilities. In mixtures containing more than 40% Hb S, polymerization takes place by the same mechanism as in solutions of Hb S alone, with no evidence for independent crystallization of Hb C. A detailed comparison of Hb S/Hb C and Hb S/Hb A mixtures with identical concentrations and proportions of Hb S show that there is no significant difference in the tendency of Hb C and Hb A to copolymerize with Hb S. In 50:50 Hb S/Hb C mixtures, polymerization is about 15 times more rapid than in 40:60 Hb S/Hb A mixtures at the same total Hb concentration. Measurements on density-fractionated erythrocytes show that SC cells contain a higher total Hb concentration and a more uniform distribution of reticulocytes compared to normal (AA) or sickle trait (AS) cells. The concentration distribution for C trait (AC) cells is much closer to that of SC cells than to AS or AA cells. It appears, therefore, that the presence of Hb C results in the SC cell beginning its life with an abnormally high Hb concentration. From these findings we conclude that both the larger proportion of Hb S and the higher intracellular Hb concentration contribute to the pathogenesis of Hb SC disease.     

Relation of haemolytic anaemia and erythrocyte-bound IgG in α- and β-thalassaemic syndromes

Abstract: There has been evidence that IgG-mediated phagocytosis plays some part in destruction of erythroid cells and subsequent anaemia in thalassaemia. In this study using direct immunofluorescence and flow cytometry, erythrocyte-bound IgG was studied in 53 and 33 β-thal/Hb E and Hb H patients, respectively. The mean percentages of IgG-positive erythrocytes in both nonsplenectomized (4.4%) and splenectomized (24%) β-thal/Hb E patients were significantly higher than that in normal subjects (1.0%); p < 0.05 and < 0.001, respectively. Splenectomized β-thal/Hb E patients had significantly higher percentages of IgG-positive erythrocytes than the nonsplenectomized patients (p < 0.001). The mean percentages of IgG-positive erythrocytes in Hb H patients were comparable to those from normal controls. However, patients with α-thal 1/Hb CS had significantly higher IgG-positive erythrocytes than those with α-thal 1/α-thal 2 (p = 0.008). Splenectomized Hb H patients also had higher IgG-positive erythrocytes than nonsplenectomized Hb H patients (p < 0.039). During haemolytic crisis in some Hb H patients, high percentages of IgG-positive erythrocytes were also demonstrated. This study demonstrates the presence of increased IgG on erythrocytes in patients with β-thal/Hb E and in those with Hb H disease following splenectomy or in haemolytic crisis.     

Comparison of hemoglobin K?ln erythrocyte membranes with malondialdehyde-reacted normal erythrocyte membranes

Splenectomized patients with hemoglobin (Hb) Koln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide- reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Koln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Koln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Koln RBC membranes and MDA- reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Koln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Koln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Koln RBCs. We conclude that Hb Koln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.     

Increased insulin binding of erythrocytes and insulin sensitivity in adrenal insufficiency

We have studied 125I-insulin binding to erythrocytes (RBC) in five patients with hypoadrenocortisolism, and compared to 17 normal subjects and in nine patients with Cushing's syndrome. In another study insulin sensitivity index (ISI) was measured by the IV insulin tolerance test in four patients with hypoadrenocortisolism (1.82 +/- 0.15 mg/dL/min), and compared to 19 normal subjects and 23 patients with Cushing's syndrome (1.56 +/- 0.1 mg/min/dL). Mean insulin binding in hypocortisolism was 17.9 +/- 0.7%, and was significantly higher (P less than .01) than in normal subjects (12.0 +/- 1.4%) and was significantly (P less than .001) decreased toward normal (11.8 +/- 1.47) during replacement therapy. Increased binding in untreated hypoadrenocortisolism was due to elevated high affinity site receptor concentration as compared to the treated patients (0.10 +/- 0.015 v 0.053 +/- 0.003 nmol/L,P less than .01). These results suggest that increased insulin binding in chronic hypoadrenocortisolism may be attributed to increased insulin binding to the receptor, which can revert to normal by replacement therapy. The role of increased insulin binding to increased insulin sensitivity in hypoadrenocortisolism is discussed.     

Human red cell age, oxygen affinity and oxygen transport

The [2,3-DPG]/[Hb] ratio and the P50 were found to be lower in the 10% denser (old) than in the 10% lighter (young) red blood cell (RBC) fractions (0.57 +/- 0.13 vs 0.96 +/- 0.13 and 23.02 +/- 0.85 vs 27.47 +/- 1.05 Torr, respectively, mean +/- SD, P less than 0.0005 for both, n = 6). The RBC aging processes appear thus to affect the RBC oxygen affinity. However, the [2,3-DPG] changes do not fully explain the drop of not fully explain the drop of P50 as measured at constant [H+], [CO2] and [HbCO]. It is therefore postulated that an additional factor is involved in the regulation of the oxygen affinity in the ageing RBC. The RBC density in 59 normal individuals matched for age (infants, adult, and aged) and for sex was found to be younger in adult females than in all other groups (P less than 0.0005), including an age-matched group of pregnant women. Correspondingly, the [2,3-DPG]/[Hb] ratio and the P50 are higher in adult females than in adult males (0.92 +/- 0.10 vs 0.82 +/- 0.09, P less than 0.009, and 29.03 +/- 1.07 vs 27.72 +/- 0.82 Torr, P less than 0.002, respectively). These data are evaluated in terms of the efficiency of the oxygen transport calculating the circulatory load required to transport a given amount of oxygen to the tissues. The results indicate that the lower oxygen affinity (due to the younger RBC population) in adult females partially compensates for their lower [Hb].     

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.     

The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology

Knowledge concerning SS (homozygous for the beta s gene) red blood cell (RBC) heterogeneity has been useful for understanding the pathophysiology of sickle cell anemia. No equivalent information exists for RBCs of the compound heterozygote for the beta s and beta c genes (SC) RBCs. These RBCs are known to be denser than most cells in normal blood and even most cells in SS blood (Fabry et al, J Clin Invest 70:1284, 1981). We have analyzed the characteristics of SC RBC heterogeneity and find that: (1) SC cells exhibit unusual morphologic features, particularly the tendency for membrane "folding" (multifolded, unifolded, and triangular shapes are all common); (2) SC RBCs containing crystals and some containing round hemoglobin (Hb) aggregates (billiard-ball cells) are detectable in circulating SC blood; (3) in contrast to normal reticulocytes, which are found mainly in a low-density RBC fraction, SC reticulocytes are found in the densest SC RBC fraction; and (4) both deoxygenation and replacement of extracellular Cl- by NO3- (both inhibitors of K:Cl cotransport) led to moderate depopulation of the dense fraction and a dramatic shift of the reticulocytes to lower density fractions. We conclude that the RBC heterogeneity of SC disease is very different from that of SS disease. The major contributions of properties introduced by HbC are "folded" RBCs, intracellular crystal formation in circulating SC cells, and apparently a very active K:Cl cotransporter that leads to unusually dense reticulocytes.     

Favism: disordered erythrocyte calcium homeostasis

The biochemical events that take place during acute hemolysis of G6PD- deficient subjects in favism are far from being elucidated. Evidence is here reported for a constantly and heavily disordered calcium homeostasis in the erythrocytes from seven favic patients. The abnormality, ie, a significantly impaired calcium ATPase activity and a parallel marked increase of intracellular calcium levels, was characteristic of the acute hemolytic crisis although unrelated to the attendant reticulocytosis. Concomitantly, a remarkable decrease of intracellular potassium was also observed. The mean +/- SD Ca2+-ATPase activity in the favic patients was 20.8 +/- 7.8 mumol Pi/g Hb/h compared with 37.2 +/- 8.5 in the matched controls represented by 12 healthy G6PD-deficient subjects (P less than .001). The mean +/- SD intraerythrocytic calcium content was 288 +/- 158 mumol/L of erythrocytes in the favic patients as compared with 22.0 +/- 8.2 in the G6PD-deficient controls (P less than .001). The intraerythrocytic potassium content was 76.6 +/- 19.3 mmol/L of erythrocytes in the favic patients and 106.6 +/- 8.2 in the G6PD-deficient controls (P less than .001). In vitro incubation of normal and G6PD-deficient erythrocytes with divicine, a pyrimidine aglycone present in fava beans and strongly implicated in the pathogenesis of favism, reproduces most of these events, including drop of calcium ATPase, increased intracellular calcium, and leakage of erythrocyte potassium.     

Molecular stability and function of hemoglobins Hasharon (alpha(2)47 (CD5)Asp----His beta 2) and Hasharon (alpha(2)47 (CD5)Asp----His delta 2)

The molecular stability and function of hemoglobin (Hb) Hasharon (alpha 2 H beta 2) and Hb Hasharon2 (alpha 2 H delta 2) were studied and compared to Hbs A, A2 and S. Hb Hasharon and Hb Hasharon2 had slightly lower P50 values than Hb A and Hb A2 but had normal responses to organic phosphates. The molecular stability of Hb Hasharon and Hb Hasharon2 (as measured by mechanical shaking and heat denaturation at 60 degrees C) were less than Hb A and Hb A2 but greater than Hb S in the oxy- and carbonmonoxy-forms. In the met-form, however, Hb Hasharon and Hb Hasharon2 were less stable than hemoglobins S, A and A2. The oxy-form of Hb Hasharon forms methemoglobin at a faster rate than Hb A and Hb S. The mechanical and heat stabilities and the rate of methemoglobin formation of oxy-Hb Hasharon were studied in the presence of sulfisoxazole. This drug increased the rate of methemoglobin formation, thus causing a further decrease in the stability of Hb Hasharon. The relationship between these laboratory findings and previously observed clinical findings associated with Hb Hasharon are discussed.     

Erythrocyte vitamin E is oxidized at a lower peroxide concentration in neonates than in adults

Erythrocytes of neonates and adults were incubated with increasing concentrations of H2O2 in the presence of a catalase inhibitor and in the absence of glucose; the pattern of oxidation of vitamin E was analyzed in relationship to that of glutathione, hemoglobin, and polyunsaturated fatty acids (PUFA), and in relationship to hemolysis. The changes of these various parameters were analyzed in function of H2O2 concentration and in relation to incubation time, and were compared in erythrocytes from neonates and adults. In the absence of H2O2, erythrocyte glutathione and tocopherol levels were similar in neonates and adults, despite fourfold lower serum vitamin E level in neonates; alpha-tocopherolquinone, methemoglobin, and malonyldialdehyde (MDA) were not detectable. At 0.375 mmol/L of H2O2, glutathione was completely oxidized. Erythrocyte alpha-tocopherol remained unchanged up to 0.75 mmol/L of H2O2, then decreased linearly, with increasing H2O2 concentrations to 10% of its initial value at 1.5 mmol/L of H2O2 in erythrocytes from neonates, whereas those from adults required 2.0 mmol/L of H2O2 (P less than .05) for the same level of oxidation. The formation of alpha-tocopherolquinone appeared inversely related to the decrease of alpha-tocopherol. The incubation time did not influence the level of vitamin E oxidation. MDA was generated autocatalytically and resulted in hemolysis at 1.5 mmol/L of H2O2 in erythrocytes from neonates and at 3.5 mmol/L of H2O2 in erythrocytes from adults (P less than .001). After four hours of incubation, MDA reached a plateau at a greater level (365 +/- 46 nmol/L) in cells of neonates than in those of adults (208 +/- 37 nmol/L/mL) (P less than .001). Hemoglobin was oxidized in the same pattern in erythrocytes of neonates and adults, and 90% of it was oxidized at 0.625 mmol/L of H2O2. In conclusion, in the experimental conditions used, oxidation of glutathione precedes that of vitamin E, and tocopherol is the last antioxidant to be consumed before the autocatalytic generation of MDA. Differences in the pattern of vitamin E oxidation, MDA generation, and hemolysis in erythrocytes from neonates and adults may be due to a lower erythrocyte vitamin E-PUFA ratio in neonates.     

A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S- Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S- Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.     

Phenotype-genotype correlation in Sicilian patients with Hb H

We studied 15 Sicilian subjects with Hb H disease correlating clinical examinations with hematological and molecular data. Seven different alpha-tha1 mutations were identified: four deletion types (--MED --CAL, -alpha3.7, -alpha4.2) and three nondeletion types (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). All the patients had a zero-gene chromosome (--MED or --CAL), while the third alpha gene was deleted (-alpha3.7, -alpha4.2) or inactive (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). In patients with the nondeletion genotype the analysis of hematological values revealed lower levels of RBC and Hb A2 and significantly higher levels of Hb H. The clinical variability was remarkable, ranging from totally asymptomatic conditions, casually diagnosed, to severe thalassemia intermedia with marked hemolytic crises, liver and spleen enlargement and the necessity for frequent transfusions. The genotype did not justify the gravity of the phenotype in every case, and the differences in clinical manifestations, also notable, are not easily explainable in subjects who apparently have the same genotype.     

Abnormal haemoglobins in the Sudan savanna of Nigeria. III. Malaria, immunoglobulins and antimalarial antibodies in sickle cell disease.

Subjects with sickle cell disease were identified in (i) a whole population sample (2742) Garki District, Kano State, Nigeria, and in (ii) the 534 infants born into the population during five years. Eleven (2.1%) newborn had Hb.SS, as was expected from gene frequency (0.146). Prevalence was maintained in the first year of life, but fell to 0.4% at one to four years and to 0.05% (one person) over the age of nine years. Antimalarial intervention for two transmission seasons was followed by an apparent but not significant decrease in Hb.SS mortality. There was one male aged about 40 years who had Hb.SC (the expected number was three). Hb.SS children were compared to normal subjects at the same age, the same village and the same survey; they had significantly less than the expected Plasmodium malariae infection (P less than 0.01) and lower than median P. falciparum densities while below five years (P less than 0.05). Over one year of age, they tended to have below average indirect fluorescent antibody (IFA) (P less than 0.01), indirect haemagglutinating antibody (IHA) (P less than 0.01) titres and number of precipitin rings (not significant) against P. falciparum antigen, and IFA against P malariae (P less than 0.01). They had above average IgM (P less than 0.05), but their IgG concentrations did not differ from normal. We conclude that (i) sickling is sufficient to protect against P. malariae in Hb.SS but not Hb.AS; (ii) sickling prevents intense P. falciparum infection in Hb.SS, as in Hb.AS; (iii) in Hb.SS, there is less antigenic stimulus and hence less antibody against P. falciparum (like Hb.AS) and P. malariae (unlike Hb.AS); (iv) although less intense, malaria is frequently fatal in Hb.SS, especially in age-group one to four years (unlike Hb.AS); (v) IgM levels are high in Hb.SS in response to frequent infections other than malaria (unlike Hb.AS).     

Selected indices of micronutrient status in adult patients with sickle cell anemia (SCA)

In 24 adults with hemoglobin SS followed at the Duke University Comprehensive Sickle Cell Center, we have studied the following nutritional parameters: reduced ascorbic acid; dehydroascorbic acid; alpha and beta carotenes; cryptoxanthin; and alpha and gamma tocopherols in whole blood, washed red blood cells, plasma, or serum. In the same population we also examined reduced glutathione (GSH) and oxidized glutathione (GSSG). Fifteen of these 24 patients also were interviewed for usual dietary intakes using a 28-day dietary history. Data obtained from patients with hemoglobin SS, sickle cell anemia (SCA) were compared to those found for seven healthy normal black adults of similar age. Plasma alpha tocopherol levels were significantly lower in SCA individuals than those of the controls (P less than 0.004). Alpha and gamma tocopherol levels in sickle RBCs were significantly higher than those from RBC suspensions of control subjects (P less than 0.007, and P less than 0.001, respectively). All serum values for carotenoids examined, specifically, beta carotene, alpha carotene, and cryptoxanthin were also markedly depressed when compared to those of healthy controls (P less than 0.001, P less than 0.002, and P less than 0.001, respectively). No other statistically significant differences were found between the two groups for any of the remaining variables, including dietary estimates. Dietary analyses suggest that dietary intakes of SCA individuals exceeded the recommended daily allowances (RDA) of all macro- and micronutrients measured, and intakes of most nutrients exceeded those of black controls interviewed. These results suggest that in individuals with SCA, several micronutrients vital to maintaining reducing capacity are present in diminished quantities in plasma/serum. These anomalies exist in SCA patients even though their intake of these micronutrients are similar to those of healthy black men and women.     

Ionic strength dependence of the polymer solubilities of deoxyhemoglobin S + C and S + A mixtures

Factors contributing to the clinical differences between sickle cell- hemoglobin C disease (SC) and the benign sickle cell trait (AS) include the higher proportion of hemoglobin (Hb) S and the higher cell Hb concentrations in SC compared with AS red cells. Reports differ, however, about whether Hb C copolymerizes more than Hb A with Hb S when measured by minimum gelling concentrations (MGCs) and polymer solubilities of the deoxy-Hb mixtures. We now show that the MGCs and solubilities of equimolar mixtures of Hb S + Hb C vary much more with the ionic strength (mu) of the solution than those of Hb S + Hb A mixtures. At mu less than or equal to 0.20, but not at mu greater than 0.25, Hb S + Hb C solubilities were significantly lower than those of Hb S + Hb A. These differences which may reflect a greater effect of the beta 6Lys+ in Hb C at lower mu, can account for the reported discrepancies. The solubility differences were similar in the presence or absence of asymmetric hybrids, and since the intratetramerically cross-linked hybrids alpha 2 beta s beta A and alpha 2 beta s beta c had similar solubilities, they did not indicate the usual mechanism, involving greater incorporation of alpha 2 beta s beta c into the polymers. The small solubility differences between the two Hb mixtures at physiologic (red cell) concentrations of Hb and 2,3- diphosphoglycerate probably play a minor role in the clinical differences between SC and AS states.     

Hematologic Profile and Lymphocyte Subpopulations in Hemoglobin SC disease: Comparison With Hemoglobin SS and Black Controls

Compared with subjects with homozygous SS disease (Hb SS), persons with hemoglobin SC (Hb SC) are known to have a more gradual loss of splenic function, a lower incidence of bacterial infections, and fewer end-organ failures. We studied hematological indices and lymphocyte subpopulations of 27 Hb SC subjects and compared them with 173 Hb SS patients and 131 black controls. Hb SC patients had higher hemoglobin levels than Hb SS subjects, lower total leukocyte, granulocyte, monocyte, and lymphocyte counts. Platelets decresed with age but not significantly, instead of incressing as among Hb SS patients. Mononuclerar cells were generally similar to controls with the exception of CD8⁺HLA-DR⁺ counts resembling Hb SS, Hematologic changes in Hb SC are limited to moderate granulocytosis in children and aduts, mild monocytosis in aduts, and increased activation of just one lymphocyte subset among those measured.