Ernest A. Turner

Nitrogen Metabolism in Sickle Cell Anemia: Free Amino Acids in Plasma and Urine

Twenty-four hour urinary levels and fasting plasma concentrations of free amino acids (AA) were evaluated in adult sickle cell anemia (HbSS) and age-matched HbAA subjects with comparable daily energy (E) and protein (N) intake. HbSS elicited significant reduction in the sum of plasma indispensable (EAA) with no change in the dispensable (NAA) amino acids, resulting in a prominent (P less than 0.01) reduction in the EAA/NAA ratio from 71% to 55%. Arg (-38%), Leu (-32%), Val (-28%) and His (-32%) were among the AA most severely affected. Despite a twofold increase in 24-hour urine volume in HbSS compared with HbAA subjects, total urinary losses of EAA and NAA were markedly reduced in the former group, with Arg (-53%) and Gly (-56%) levels most prominently affected. Compared with HbAA controls, HbSS subjects showed a three-fourfold increase in 24-hour urinary orotate excretion that had no relationship to amount of N intake. The results indicated that adult subjects with HbSS, who consumed adequate N and E as per the RDA for healthy individuals, behaved like normal HbAA controls on a low protein diet. There was evidence that the HbSS subject might be in a precarious state with respect to sufficiency of several amino acids, particularly L-Arg, which is now classified as conditionally indispensable for the human.

Alterations in basal nutrient metabolism increase resting energy expenditure in sickle cell disease

Basal rates of whole body protein, glucose, and lipid metabolism and resting energy expenditure (REE) were measured in eight African-American sickle cell disease (SCD) patients and in six African-American controls. Catheters were placed 1) in an antecubital vein for stable isotope infusion and 2) in a heated hand vein for arterialized venous blood. Breath and blood were collected during the last 30 min of the 2.5-h study, and REE was measured by indirect calorimetry. REE [128 ± 5 vs. 111 ± 1 kJ ⋅ kg fat-free mass (FFM)-1 ⋅ day-1; P < 0.05 vs. controls] was 15% greater in the SCD patients. Whole body protein breakdown (5.0 ± 0.3 vs. 3.8 ± 0.2 mg ⋅ kg FFM-1 ⋅ min-1; P < 0.05 vs. controls) and protein synthesis (4.4 ± 0.3 vs. 3.2 ± 0.2 mg ⋅ kg FFM-1 ⋅ min-1; P< 0.05 vs. controls) were 32 and 38% greater, respectively, in the SCD patients, but whole body amino acid oxidation was similar (0.58 ± 0.03 vs. 0.66 ± 0.03 mg ⋅ kg FFM-1 ⋅ min-1). Measures of whole body glucose and lipid metabolism were not significantly different between the groups. The additional energy required for the greater rates of whole body protein breakdown and synthesis caused by SCD contributes significantly to the observed increase in REE, suggesting that dietary energy and protein requirements are enhanced in SCD patients.Basal rates of whole body protein, glucose, and lipid metabolism and resting energy expenditure (REE) were measured in eight African-American sickle cell disease (SCD) patients and in six African-American controls. Catheters were placed 1) in an antecubital vein for stable isotope infusion and 2) in a heated hand vein for arterialized venous blood. Breath and blood were collected during the last 30 min of the 2.5-h study, and REE was measured by indirect calorimetry. REE [128 +/- 5 vs. 111 +/- 1 kJ.kg fat-free mass (FFM)-1.day-1; P < 0.05 vs. controls] was 15% greater in the SCD patients. Whole body protein breakdown (5.0 +/- 0.3 vs. 3.8 +/- 0.2 mg.kg FFM-1.min-1; P < 0.05 vs. controls) and protein synthesis (4.4 +/- 0.3 vs. 3.2 +/- 0.2 mg.kg FFM-1.min-1; P < 0.05 vs. controls) were 32 and 38% greater, respectively, in the SCD patients, but whole body amino acid oxidation was similar (0.58 +/- 0.03 vs. 0.66 +/- 0.03 mg.kg FFM-1.min-1). Measures of whole body glucose and lipid metabolism were not significantly different between the groups. The additional energy required for the greater rates of whole body protein breakdown and synthesis caused by SCD contributes significantly to the observed increase in REE, suggesting that dietary energy and protein requirements are enhanced in SCD patients.

In vitro effects of NIPRISAN (Nix-0699): a naturally occurring, potent antisickling agent.

Summary. Among the various potential antisickling agents tested, hydroxyurea (HU) has been the most effective compound used for the treatment of patients with sickle cell disease (SCD). Although HU is effective in many patients, not all patients respond to this drug. In addition, some patients reveal adverse effects, including myelosuppression. In an attempt to find other effective agents with less adverse effects, we investigated the antisickling effect of NIPRISAN (Nix‐0699). We found that Nix‐0699, an ethanol/water extract from indigenous plants, has a strong antisickling effect. The concentration of Nix‐0699 required to inhibit 50% of erythrocyte sickling was about 0·05 mg/ml. As for the kinetics of polymerization, addition of 0·05 μg/ml Nix‐0699 caused a sixfold prolongation of the delay time prior to deoxy‐Hb S polymerization when compared with that of untreated Hb S samples. The solubility of deoxy‐Hb S significantly increased upon treatment with Nix‐0699. Analysis of the effect of Nix‐0699 on the Hb S oxygen affinity indicated that the drug slightly shifted the oxygen‐dissociation curve of Hb S toward the left without any apparent change in the Hill coefficient. These results suggest that the antisickling properties of Nix‐0699 may involve direct interaction with Hb molecules. Incubation of red blood cell (RBC) suspensions with various concentrations of Nix‐0699 did not dehydrate RBCs, cause haemolysis, increase the amount of denatured Hb, nor form met‐Hb. In view of the outcome of this study, Nix‐0699 may be a promising option for the treatment of patients with SCD.

Niprisan (Nix-0699) improves the survival rates of transgenic sickle cell mice under acute severe hypoxic conditions

Summary. The substitution of glutamic acid by valine at the sixth position of the beta‐globins of haemoglobin S (Hb S) causes a drastic reduction in the solubility of the deoxy form of Hb S. Under hypoxic conditions, deoxy‐Hb S molecules polymerize inside the cells, forming rigid, sickled cells. We studied the effect of Niprisan (Nix‐0699), a naturally occurring antisickling agent, on the survival of transgenic (Tg) sickle mice under severe acute hypoxic conditions (60 min). Before hypoxia exposure, the mice were treated by gavage once daily for 7 d with 0 mg/kg (n = 10), 10 mg/kg (n = 5), 50 mg/kg (n = 5), 300 mg/kg (n = 4) or 500 mg/kg (n = 5) of Nix‐0699. The mean survival times of the untreated and treated mice were 10, 25, 39, 55 or 60 min respectively. The percentage of sickled cells in the venous blood of the treated mice was lower than that in control mice and was dose dependent. Histological examination of the lungs of the control mice showed entrapment of massive numbers of sickled cells in the alveolar capillaries, although the degree of such entrapment decreased with the increased dose of Nix‐0699. Nix‐0699 may be a promising option for the treatment and management of patients with sickle cell disease.

Enhancement of hemoglobin and F-cell production by targeting growth inhibition and differentiation of K562 cells with ribonucleotide reductase inhibitors (didox and trimidox) in combination with streptozotocin.

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F‐cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation. Of the RRIs currently in use, hydroxyurea is the most effective agent for Hb F induction. We have examined the capacity of two novel RRIs, didox (DI) and trimidox (TRI), in combination with streptozotocin (STZ), to induce hemoglobin and F‐cell production. The K562 cells were cultured with different concentrations of didox‐STZ or trimidox‐STZ at a fixed molar ratio of 3:1 and 1:5 for 96 hr, respectively. At pre‐determined time intervals, aliquots of cells were obtained and total hemoglobin (benzidine positive) levels, number of F‐cells, and Hb F were determined by the differential staining technique, fetal hemoglobin assay kit, and fluorescence cytometry respectively. The effect of combined drug treatment on the growth of K562 cells was examined by isobologram analysis. Our results indicate that a synergistic growth‐inhibitory differentiation effect occurred when didox or trimidox was used in combination with STZ on K562 cells. There was an increase in the number of both benzidine‐positive normoblasts and F‐cells, accompanied by morphologic appearances typical of erythroid maturation. On day 4, the number of benzidine‐positive cells showed a 6–9‐fold increase and the number of F‐cells was between 2.5‐ and 5.7‐fold higher than the respective controls. Based upon these results, treatment with a ribonucleotide reductase inhibitor, such as didox or trimidox, in combination with STZ, might offer an additional promising option in sickle cell disease therapy. Am. J. Hematol. 63:176–183, 2000.

New isocratic high-performance liquid chromatographic procedure to assay the anti-sickling compound hydroxyurea in plasma with ultraviolet detection

A new procedure using high-performance liquid chromatography (HPLC) with ultraviolet detection to assay hydroxyurea (HU) levels in plasma has been developed. The drug was isolated from plasma by a direct deproteinization process with sulfosalicylic acid. Following neutralization of the acidic supernatant, an aliquot was loaded onto an Aminex HPX-72S column (300x7.8 mm). Chromatography was performed at 55 degrees C using a mobile phase consisting of acetonitrile-0.025 M ammonium sulfate buffer (pH 8.5) including 0.1% triethylamine, 0.01 M sodium sulfate, and 5 mM sodium heptane sulfonate. The UV absorbance of effluent was monitored at 214 nm. A flow-rate of 0.8 ml/min was used for analyzing HU in both human and mouse plasma. Under these conditions, the drug eluted at 12.6 min. The assay possessed linearity up to 425 microg/ml, with a lower limit of quantitation of 3.32+/-0.0004 microg/ml (mean+/-S.D., n=10). Intra-day and inter-day coefficients of variation were less than 8.5% and 8.7% respectively. Absolute differences were less than 7.4%. The method has been employed in clinical studies and the sensitivity of the assay was shown to be adequate for characterizing the plasma pharmacokinetics of HU in mice. In conclusion, the procedure described herein could be ideally suited for therapeutic monitoring of hydroxyurea.

Plasma leptin association with body composition and energy expenditure in sickle cell disease.

Objective: To examine the association between fasting plasma leptin concentrations and the hypercatabolic state observed in sickle cell disease (SCD). Methods: Plasma leptin concentration and resting energy expenditure (REE) were measured in 37 SCD patients (10 men, 12 boys 14 to 18 years-old, seven women, and eight girls 14 to 18 year-old) and in 37 age, gender and fat mass (FM) matched controls. Body composition was measured hydrostatically, REE by whole room-indirect calorimeter, and plasma leptin using an RIA kit. Results: Plasma leptin concentration and leptin normalized for body fat (ng/dL*kg FM−1) were significantly lower in SCD patients than in non-SCD controls (4.00±3.23 vs. 9.94±14.69, p=0.021 and 0.406±0.260 vs. 0.643±0.561, p=0.024, respectively). A positive linear association between log plasma leptin and FM was observed in both males and females, adjusting for age and SCD status. The strength of this association was greater in females compared with males (slope=0.699 and 0.382 log ng/mL per 10 kg FM, respectively; p=0.013). SCD patients on average demonstrated a higher REE, adjusting for FFM (p<0.0001). Log plasma leptin and FM were not statistically significant predictors of REE after adjustment for FFM and SCD. Conclusions: Once corrected for body composition, mean plasma leptin concentration was significantly lower among female SCD patients than among non-SCD matched controls. Although REE was higher in SCD patients, there is no simple association between leptin and REE in SCD.

Hydroxyurea‐induced oxidative damage of normal and sickle cell hemoglobins in vitro: Amelioration by radical scavengers

Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes. In vitro exposure of human blood to various concentrations of HU at predetermined time intervals induced a progressive dose‐dependent oxidation (MetHb formation) of both adult (Hb AA) and sickle (Hb SS) hemoglobins. The oxidative effect of HU on Hb SS was 3 times greater than its effect on Hb AA. Similar but less profound changes were observed in H2O2‐treated samples. Hb F was, however, observed to be relatively resistant to HU‐induced oxidative damage. A substantial protective effect of Hb by α‐tocopherol, ascorbic acid, and D‐mannitol was observed during pretreatment of Hb AA and Hb SS blood samples. Analyses of the hemoglobins and their globin chain components by high‐performance liquid chromatography revealed a considerable protective effect by these free radical scavengers. These results indicate that the HU‐induced damage of hemoglobin and their component globin chains can be reduced by radical scavengers. J. Clin. Lab. Anal. 15:1–7, 2001.

Hydroxyurea-induced denaturation of normal and sickle cell hemoglobins in vitro

Use of hydroxyurea (HU) to treat sickle cell disease is usually associated with increments in fetal hemoglobin (Hb F) production; however, in vitro studies show that HU may also induce hemoglobin denaturation. Whole blood samples from Hb AA, Hb AS, and Hb SS patients were treated in vitro with 100, 150, 200, 250, and 300 μg/mL HU, incubated at 30°C for up to 12 days, and analyzed by high‐performance liquid chromatography (HPLC). Hb AA levels show decrements of 91 to 14% with 100 μg/mL and 89 to 4% with 150 μg/mL after 12 days; 86 to 2% with 200 μg/mL after 10 days; 86 to 8% with 250 and 300 μg/mL after 8 days. Similar treatment and incubation times for Hb AS whole blood demonstrate that HU equally degrades the A and S components of Hb AS. A comparable approach for Hb SS whole blood samples, using a 300 μg/mL HU treatment, showed a hemoglobin denaturing pattern that went from 93% to 1% after 12 days. Globin chain analysis of these samples by reverse‐phase HPLC showed that the denaturing effects occur mostly on the β‐globin chain. J. Clin. Lab. Anal. 11:208–213, 1997.