Elevated urinary levels of thromboxane and prostacyclin metabolities in sickle cell disease reflects activated platelets in the circulation.

There is evidence for increased factor VII turnover and the associated increased thrombin generation and fibrinolytic activities in sickle cell disease (SCD) that may affect in vivo platelet and endothelial cell reactivity. We studied the release of specific eicosanoids that are indicative of in vivo platelet activation and endothelial cell injury. The circulating and urinary levels of 2,3-dinor thromboxane B2(2,3-dinor-TxB2),TxB2,PGI2 [as 6-keto-PGF1 alpha], and PGE2 were measured in 15HbSS patients, eight HbAA non-haemolytic anaemic individuals and 12 healthy HbAA controls using specific RIAs. The mean urinary 2,3-dinor-TxB2 in the HbSS patients was significantly higher than in both the healthy HbAA and the anaemic controls. 6-keto-PGF1 alpha was undetected in the urines of the healthy HbAA controls, but was measured insignificant amounts in the HbSS and the HbAA anaemic patients. The urinary concentrations of PGE2 and TxB2 in HbSS patients' samples were also significantly higher than those of both control groups (P < 0.05). PGE2 and TxB2 levels were below the detection limit in the plasmas of the HbAA subjects, but were measurable in the HbSS and HbAA anaemic plasmas. The plasma level of 6-keto-PGF1 alpha in the HbSS patients was also significantly higher than in the control groups. The data indicates a persistent inflammatory process in the HbSS patients, and is consistent with the hypothesis that there is platelet and endothelial cell activation in SCD.

IgG subclass alterations in adult asthma.

Immunoglobulin levels were measured in serum samples of 12 black adult non-smoking asthmatic patients, 11 females and 1 male, and compared with 15 age-, sex-matched normal controls. Their total IgG, IgA and IgM levels were within the normal range. However, on quantitation of subclasses, IgG1 levels were significantly above normal, while IgG2 and IgG3 levels were significantly lower than those of controls. No significant differences were found between the two groups when IgG4 levels were compared. These studies as well as those of others suggest that immunoglobulin administration, particularly of individual subclasses, might prove to be a beneficial addition in the management of this condition.

Selenium and glutathione peroxidase levels in sickle cell anemia.

Levels of plasma selenium (Se) and glutathione peroxidase were measured in 20 sickle cell anemia (SCA) patients not in crisis and in 14 nonanemic control subjects. The results show that the levels of Se and glutathione peroxidase were significantly (p less than 0.005) lower than those of controls in both plasma and whole blood. These data are consistent with the previous reports that there is increased oxidative stress in SCA. Low blood Se levels and glutathione peroxidase activity observed in this research suggest that a weakened antioxidant potential may be associated with SCA patients. The low Se status in SCA patients may also affect the phenotypic expression of these patients.

Alterations in IgG subclasses in acquired immune deficiency syndrome.

Decreased IgG subclass levels in pyogenic infections and immunocompromised situations have been described. A study was made to determine IgG subclass levels in four groups of 68 Hispanic patients. The first group consisted of 25 terminal patients with AIDS, the second group of 20 i.v. drug abusers, and the third group of eight hospital patients with neither a diagnosis of AIDS/ARC nor a history of i.v. drug abuse. IgG subclass levels of these 53 cases were compared with those of a fourth group of 15 normal controls. The total IgG, IgA, and IgM levels as well as the four IgG subclass concentrations were measured by radial immunodiffusion using appropriate standards and specific antisera. The first two groups had similar values, with an average IgG level of 10.37 g/liter; IgA, 2.68; and IgM, 1.78; subclass levels were IgG1, 6.68 g/liter; IgG2, 2.77; IgG3, 0.34; and IgG4, 0.68. These were significantly lower than those of controls, except for IgG4. Determination of minor subclasses may offer some possibilities for immunomodulation and therapy and could be useful in terms of prognosis.

Low serum levels of carotenoids in sickle cell anemia.

Serum carotenoids, tocopherols and retinol were analyzed in patients with sickle cell anemia (SCA) and in control subjects. The data show the following: the serum levels of the major carotenoids, alpha and beta carotene, cryptoxanthin, lycopene lutein, alpha tocopherol and retinol were significantly lower in SCA patients. These findings reflect an additional abnormality of the antioxidant system in SCA patients. Gamma tocopherol, on the other hand, was significantly elevated, consistent with the previously reported reciprocal relationship between serum alpha and gamma tocopherols. Thus, these data taken together with the earlier findings on the lower levels of plasma alpha tocopherol and ascorbic acid suggest that the entire antioxidant system may be compromised in SCA patients. This may contribute in part to the phenotypic expression of the condition.

Depressed plasma pyridoxal phosphate concentrations in adult asthmatics.

In 15 adult patients with bronchial asthma, plasma and erythrocyte pyridoxal phosphate (PLP) concentrations were significantly lower than in 16 controls (P less than 0.0001 and P less than 0.005, respectively). Oral supplementation of seven asthmatics with 50 mg pyridoxine as pyridoxine X HC1 twice daily failed to produce a sustained elevation of PLP in either the plasma or erythrocytes. However, all subjects reported a dramatic decrease in frequency and severity of wheezing or asthmatic attacks while taking the supplement. The reasons for the failure of a uniform elevation in plasma and erythrocyte PLP concentration and for the apparent beneficial effects of pyridoxine supplementation on the asthmatic symptoms of the patients are unknown at present.

Compartmentalization of iron in sickle cell anemia--an autopsy study.

In autopsy studies of two sickle cell anemia (SCA) patients, iron was totally absent from all organs in one case in which the serum ferritin had been low, while the other patient demonstrated hepatic, splenic, and renal siderosis but no iron stores in the bone marrow. Serum ferritin had been very high in this case. These findings suggest that in SCA bone marrow biopsy may be an unreliable means to determine iron stores, but rather the ferritin level should be used.

IgG2 deficiency in sickle cell anaemia.

8 patients with known sickle cell anaemia were studied immunologically. The concentrations of the main immunoglobulin classes, IgG and IgA, were significantly higher than the levels in 11 normal age- and sex-matched black subjects (P less than 0.01). IgM levels were not significantly different in the two groups. There was a heterogeneity in the interaction of the IgG subclasses with Protein A, with low levels of IgG2. The IgG2:IgG1 ratios varied from 1:3.8 to 1:6 (normals 1:3). In 4 patients the absolute levels of IgG2 as measured by radial immunodiffusion were lower than normal, thus confirming the chromatographic ratios. Since specific antibody is often restricted to a single subclass, the levels of IgG subclasses may be related to recurrent bacterial infections in these patients.

Apparent vitamin B6 deficiency in sickle cell anemia.

In 16 patients with sickle cell anemia, plasma pyridoxal phosphate (PLP) concentrations were significantly lower than in 16 controls (p less than 0.0001) whereas sickle cell anemia erythrocyte PLP concentrations were significantly elevated (p less than 0.0005), possibly reflecting a greater affinity of PLP to the sickle Hb beta chain compared to the normal beta chain. Oral supplementation of five patients with 50 mg pyridoxine twice daily for 2 months resulted in increased plasma and erythrocyte PLP levels and a slight, but not significant, increase in erythrocyte cell number, Hb, concentration, and hematocrit. In one subject there was also a reduction in the frequency and duration of painful crises and a virtual elimination of hospitalizations for the treatment of the painful crises. Since pyridoxal and PLP have been shown to have antisickling properties in vitro, these studies suggest that pyridoxine supplementation may also be of therapeutic benefit in vivo in sickle cell anemia.

A family study of IgG subclasses in sickle cell anemia.

Three siblings with sickle cell anemia were studied immunologically and hematologically. Their patterns of Protein A-Sepharose chromatography distribution showed considerable heterogeneity, particularly with respect to the IgG2 and IgG3 subclasses, even though their hematological make up was similar. An attempt was made to correlate their IgG2: IgG1 subclass ratios with their clinical history of recurrent bacterial infections, as well as a possible compensatory IgG3 heterogeneity.

Polyamines and membrane proteins in sickle cell disease.

The polyamines, putrescine, spermidine, and spermine have been implicated in cellular growth and membrane stabilization. We have shown that RBC stroma and lysate polyamines are significantly elevated in sickle cell disease (SS and SC) relative to controls (AA). The crude spectrin fraction of the red cell stroma has associated with it a large fraction of the total stroma polyamines, suggesting that the polyamines in the stroma are preferentially bound to spectrin. The high levels of polyamines associated with the spectrin of young and ISC's relative to normal may contribute to an alteration of the physical-chemical properties of the sickle RBC membrane. Spermine levels were high in the SS lysates but not detectable in SC or AA lysates. Levels in SC patients were intermediate. In preliminary experiments using washed red cells obtained from SS and AA subjects, there was extensive incorporation of 14C putrescine into red cell protein. Labelled N-(gamma-glutamyl) putrescine was isolated, consistent with covalent binding of the polyamines to protein.

Polyamines and globin binding in sickle cell disease.

Erythrocytes obtained from patients with sickle cell anemia contain five to ten times more spermine than controls. The preferential binding of beta s globin to red cell stroma may be mediated by spermine and may account in part for the membrane abnormalities found in sickle cell disorders.

Glutathione peroxidase activity in whole blood of patients with sickle cell anaemia.

Whole blood glutathione peroxidase was found significantly increased relative to controls in a group of 21 black patients with sickle cell anaemia. One control group consisted of 15 normal black subjects. A second control group, consisting of 21 black patients with various abnormal haemoglobins including alpha-thalassaemia, also showed a tendency to enhanced glutathione peroxidase activity, confirming previous reports that elevated glutathione peroxidase levels are secondary to a variety of haemolytic conditions, rather than typical of sickle cell anaemia.

A decrease in irreversibly sickled erythrocytes in sicle cell anemia patients given vitamin E.

Patients with sickle cell anemia were given 450 IU of vitamin E (as alpha-tocopherol) per day for 6 to 35 weeks. Plasma tocopherol levels increased from 0.7 +/- 0.2 mg/g lipid pretreatment, to 2.3 +/- 0.3 mg/g lipid. The percentage of circulating irreversibly sickled red cells decreased from 25 +/- 3% pretreatment to 11 +/- 1% after vitamin E administration (P less than 0.001). The percentage of irreversibly sickled red cells remained below pretreatment levels as long as the vitamin was administered (up to 35 weeks). The biochemical and clinical implications of these observations are discussed.

Preferential binding of beta C relative to beta S globin to stroma in hemoglobin SC disease.

Globin synthesis was measured in blood samples of four patients with hemoglobin SC Disease. In the stroma-free supernatant, globin synthesis was balanced with alpha/(beta S + beta C) = 1. In the exhaustively washed stroma, there was a preferential binding of beta chains to stroma, similar to that reported for Sickle Cell Anemia. However, while the beta S/beta C ratio was balanced in the supernatant (0.8-0.9), the beta S/beta C ratio of the stroma varied from 0.2 to 0.4. Therefore, beta C is bound to stroma in preference to beta S in hemoglobin SC disease. These findings are consistent with the concept that binding of globin to stroma is related to the electrophoretic charge of the hemoglobin.

Heterogeneity of DNA fragments associated with the sickle-globin gene.

We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.