Isolation of Plasmodium berghei by hemolysin lysis of infected erythrocytes and evidence for a parasite hexokinase.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.

Erythrocyte calcium abnormalities and the clinical severity of sickling disorders.

We studied erythrocyte calcium levels and uptake in a group of patients with sickle haemoglobinopathies of different clinical severity in an attempt to relate these measurements to the production of irreversibly sickled cells and disease severity. Erythrocyte calcium levels were measured by atomic absorption spectroscopy and calcium uptake by isotopic means. In sickle cell anaemia, erythrocyte calcium content was elevated and the uptake of isotopic calcium increased under both oxygenated and deoxygenated conditions. There was a direct correlation between the numbers of irreversibly sickled cells and calcium uptake and an inverse relationship between calcium uptake and red cell potassium level. The clinical course of disease was milder in patients with high fetal haemoglobin levels, but there was no relationship between clinical course and calcium levels, calcium flux or irreversibly sickled cells. Our observations suggest that calcium accumulation and irreversibly sickled cell formation are related processes. The absence of good correlation between various biochemical and clinical parameters emphasizes the complexity of factors which modify the clinical course of this disorder.

Association between cholesterol and 2,3-diphosphoglycerate in genetically selected hooded rat lines.

We have developed two strains of hooded rats with differing erythrocyte oxygen affinities by selection on red cell 2,3-diphosphoglycerate levels. Genetic studies have shown that these strains differ at one DPG-level-determining locus. This article reports the results of a study which involved measurement of plasma cholesterol levels in rats from the strains and the F2 progeny of strain intercrosses. Low-DPG strain rats, with high oxygen affinity, had significantly higher mean cholesterol levels than High-DPG rats. Animals from the extremes of the F2 distribution of DPG levels showed similar, significantly different mean cholesterol levels, indicating that the negative association between DPG and cholesterol levels in strain rats was not due to inadvertent fixation of unrelated genes during selection on DPG. The possibility is discussed that high oxygen affinity, brought about by low DPG levels, may be causative in increasing cholesterol levels.

Pharmacologic stimulation of erythrocyte 2,3-diphosphoglycerate production in vivo.

The ability of phosphate alone and in combination with sodium bicarbonate, vitamin C and pyruvate to elevate 2,3-diphosphoglycerate (DPG) levels was examined in normal human subjects in vivo. Statistically significant increases in DPG levels were noted (P less than .001) for all regimens pooled. Average rise in DPG levels reached 6% above pretreatment levels after 2 to 3 days of treatment. No changes in DPG levels were detectable in a group of control subjects over the same time period. Variation among pharmacologic trials and among subjects within trials in the actual magnitude of DPG build-up was marked. Significant differences could not be detected among the pharmacologic regimens in their ability to produce DPG increases. Previous reports suggested the use of these agents to stimulate red cell glycolysis but it seems that their effects and the mechanisms of glycolytic control in vivo are considerably more complex than has been previously suggested.

The effects of zinc on the morphology of sickle red blood cell ghosts as observed by scanning electron microscopy.

The objective of these studies is to define the role of zinc in RBC ghost morphology specifically in comparison to the echinocytogenic effects of calcium. Erythrocyte ghosts were prepared from patients with sickle cell anemia, treated with zinc and/or calcium and the resulting cell morphology studied by scanning electron microscopy. Our results showed that zinc consistently and significantly antagonized the echinocytogenic effect of calcium. A simple log linear statistical model was used to evaluate the data and supported this conclusion. We discuss the potential significance of these findings to the effect of zinc treatment on patients with sickle cell anemia.

Effects of ascorbic acid on glucose-6-phosphate dehydrogenase-deficient erythrocytes: studies in an animal model.

There is little information available concerning the possible effects of ascorbic acid on glucose-6-phosphate dehydrogenase (G-6-PD) -deficient erythrocytes. We therefore studied the survival of G-6-PD-deficient cells, incubated with ascorbic acid, in rats with partial reticuloendothelial and complement blockade. Animals were pretreated with ethyl palmitate and cobra venom factor. They were then transfused with 51Cr-labeled erythrocytes which had been incubated in the presence or absence of ascorbic acid. G-6-PD-deficient cells, incubated with ascorbic acid, had impaired survival when compared to that of controls. G-6-PD-deficient cells, in the absence of ascorbic acid, had survival times equal to those of normal control erythrocytes. At the concentrations used, ascorbic acid did not appear to alter the survival of normal cells. Ascorbic acid causes premature loss of G-6-PD-deficient human erythrocytes in this rat model. On the basis of previous clinical and experimental observations, as well as this study, caution should be used when administering ascorbic acid in large doses to individuals who might be G-6-PD deficient.

Hexokinase of the adult dog heartworm, Dirofilaria immitis.

1. Dirofilaria immitis hexokinase was relatively heat stable and had a pH optimum range between 7.8 and 8.2. 2. Mean Vmax was 0.40 +/- 0.10 (S.D.) mumole/min/100 mg of worm. 3. Mean Km values were 0.32 mM for glucose, 0.86 mM for fructose and 0.39 mM for ATP. 4. Glucose-6-phosphate was not a strong product inhibitor. 5. Starch gel electrophoresis demonstrated at least three isozymes.

Zinc in the treatment of homozygous sickle cell anemia: studies in an animal model.

We have studied the effects of zinc on the 51Cr survival of red blood cells (RBC) from patients with homozygous sickle cell anemia (SCA) using an animal model in which the RBC were transfused into specially prepared rats. The slope (lambdas) of a standard 51Cr RBC survival curve was used as a measure of the rate of RBC sequestration. The effects of intravenous zinc were of considerable therapeutic interest from the standpoint of setting guidelines for effective blood levels of zinc in patients. SCA RBC were transfused into rats whose plasma zinc levels had been raised 3-6 times above normal (300-600 mug/100 ml) by prior iv injection of zinc acetate; in three experiments the mean lambdas in zinc-treated animals breathing 15-16% oxygen was significantly lower (meaning lessened sequestration and greater survival) than saline-treated controls. A possible explanation for the requirement to lower ambient oxygen tension in order to see this zinc effect is discussed. We have further observed an increased mean lambdas for RBC from 10 SCA patients compared to 4 normal controls (0.134 vs 0.030; t = 2.8, p less than 0.01). The lambdas values are quite patient specific (4 patients studied; F = 18.2, P = 0.002). In vitro pretransfusion treatment of SCA RBC with 1.5 mM zinc resulted in a significant increase in hemoglobin oxygen affinity and a marked reduction in lambdas (0.073 vs 0.120; t = 4.5, p less than 0.01). The mean lambdas was not affected by in vitro 0.3 mM zinc treatment; this level did not change hemoglobin oxygen affinity. We conclude that systemic zinc therapy in the animal model described, at plasma levels only slightly higher than those presently obtained in patients, prolongs SCA RBC survival. This animal model is a sensitive measure of the sicklability of SCA RBC and is useful in the testing of in vitro and in vivo antisickling agents.

Zinc deficiency in sickle cell disease.

Clinical similarities between patients with sickle cell anemia and zinc-deficient subjects suggested a secondary zinc deficiency in sickle cell anemia. Zinc was assayed in various biological fluids and tissues by atomic absorption spectrophotometry. Zinc in the plasma, erythrocytes, and hair was decreased and urinary zinc excretion was increased in anemia patients as compared to controls. Erythrocyte zinc and daily urinary zinc excretion were inversely correlated in the anemia patients (r equal to minus .63, P smaller than 0.05), suggesting that hyperzincuria may have caused zinc deficiency in these patients. Carbonic anhydrase, a zinc metalloenzyme, correlated significantly with erythrocyte zinc (r equal to plus 0.94, P smaller than 0.001). Plasma RNase activity was significantly greater in anemia subjects than in controls. We administered zinc sulfate, 660 mg per day, orally, to seven men and two women with sickle cell anemia. Two 17-year-old males gained 5 cm and 7 cm in height during 49 and 42 weeks of zinc therapy, respectively. All but one patient gained weight (0.5 kg to 4.1 kg). Five of the males showed increased growth of pubic, axillary, facial, and body hair, and in one a leg ulcer healed in six weeks on zinc and in two others some benefit of zinc therapy on healing of ulcers was noted.