Ernst R. Jaffé

Hereditary nonspherocytic hemolytic disease associated with an altered phospholipid composition of the erythrocytes

A hemolytic disorder with mild hyperbilirubinemia and reticulocytosis of 6 to 15% was documented in eight members of a large family from the Dominican Republic and was presumed to be present in eight other members. The disorder appeared to be inherited as an autosomal dominant characteristic. Analysis of phospholipids by quantitative thinlayer chromatography revealed a distinct increase in phosphatidyl choline (lecithin) to 35.5 +/- SD 1.3% of the total (normal: 28.2 +/- 1.4%) in erythrocytes of affected members of the family, but not in the cells of unaffected relatives. The alteration appeared to constitute an absolute increase in lecithin content, rather than a decrease in other phospholipids. Erythrocytes from patients with other varieties of hereditary hemolytic disorders and comparable levels of reticulocytosis had normal phospholipid compositions. Plasma lipids of six affected members of the family were not unusual with respect to total lipid weight, total phospholipid, and cholesterol. Three patients with liver disease and jaundice were found to have marked increases in the lecithin content of the erythrocytes, but they also had extremely high plasma levels of total lipid, phospholipids, and cholesterol. Osmotic fragility of the erythrocytes of affected patients was decreased and the increase in fragility after incubation for 24 hr was less than that observed with normal erythrocytes. Autohemolysis after 48 hr was slightly increased and was corrected to nearly normal by the addition of glucose. The activities of 15 enzymes of the erythrocytes of the propositus were normal or elevated and the adenosine triphosphate content was normal. An abnormal hemoglobin could not be demonstrated. The life span of isologous erythrocytes in the propositus was reduced, but homologous erythrocytes survived normally.A causal relationship between the altered phospholipid composition and the hemolytic disorder has not been established.

THE REDUCTION OF METHEMOGLOBIN IN HUMAN ERYTHROCYTES INCUBATED WITH PURINE NUCLEOSIDES

This study is concerned with the effect of purine nucleosides and related compounds on the reduction of methemoglobin to hemoglobin in human erythrocytes in vitro. Other investigators have reported that mammalian erythrocytes can reduce methemoglobin to hemoglobin when the cells are incubated with glucose (2), lactate (3), glyceraldehyde, fructose (4), fumaric acid, malic acid (5), mannose, galactose (6), formaldehyde (7) and other aliphatic and aromatic aldehydes (8). Previous studies in this and other laboratories have demonstrated that normal human erythrocytes can metabolize the pentose moiety of certain purine nucleosides to lactic acid in vitro (9, 10). This metabolic activity is associated with enhanced resistance to osmotic stress (11, 12), increased organic phosphate esters (11), retention of potassium (11) and extrusion of sodium (13), maintenance of reduced glutathione (14, 15) and some prolongation of viability of erythrocytes stored in vitro (11, 16, 17). In the present investigation it was observed that normal human erythrocytes in which 60 to 80 per cent of the hemoglobin was chemically oxidized in vitro could reduce a considerable portion of the methemoglobin to hemoglobin when incubated with certain purine nucleosides and sugars. This effect was associated with active metabolism of the compounds by the cells. The metabolism of these compounds and their effects on the reduction of methemoglobin in erythrocytes obtained from a patient with congenital methemoglobinemia were also studied.

Electrophoretic and functional variants of NADH-methemoglobin reductase in hereditary methemoglobinemia

The electrophoretic mobility and activity of NADH-methemoglobin reductase in erythrocytes of patients with hereditary methemoglobinemia, obligatory heterozygotes, and normal subjects were examined. Six distinct electrophoretic variants were found in studies of erythrocytes from members of ten different families. Five variants (Boston Slow, Duarte, Princeton, Puerto Rico, and California) were associated with significant methemoglobinemia and moderate to marked decreases in enzymic activity. Precise correlations between levels of NADH-methemoglobin reductase activity, electrophoretic mobility, and clinical severity of methemoglobinemia, however, could not be drawn. One variant (Boston Fast) was associated with almost normal activity and very minimal methemoglobinemia. Nine members from three generations of two Italian families were found to have two bands with NADH-methemoglobin reductase activity in their erythrocytes, one with normal mobility and one with a mobility identical with that of Boston Fast. No functional or clinical impairment could be attributed to this abnormality. The observations made in this investigation were consistent with an autosomal recessive mode of inheritance of multiple alleles for NADH-methemoglobin reductase. As has been shown to be true for hemoglobin and glucose-6-phosphate dehydrogenase, multiple aberrations in the NADH-methemoglobin reductase of human erythrocytes apparently exist, some with and some without functional consequences. Two bands with NADPH-methemoglobin reductase activity with electrophoretic mobilities distinct from those of the NADH-methemoglobin reductase were found in human erythrocytes. These bands were normal in hemolysates of erythrocytes from patients with hereditary methemoglobinemia, but were absent from the hemolysate of erythrocytes deficient in NADPH-methemoglobin reductase activity. These latter erythrocytes, however, contained normal concentrations of methemoglobin and had a normal ability to reduce methemoglobin in vitro. These observations were most consistent with the thesis that the NADH-methemoglobin reductase, distinct from any NADPH-methemoglobin reductase, was the major system responsible for the reduction of methemoglobin to hemoglobin in human erythrocytes.

The Effects of Nucleosides On the Resistance of Normal Human Erythrocytes to Osmotic lysis

The investigations reported in this paper proceeded on the hypothesis that maintenance of the structural integrity of the human erythrocyte is dependent on continued production and utilization of energy by the cell. In an attempt to test this hypothesis the susceptibility of fresh human erythrocytes to osmotic lysis was studied in terms of the influence of various compounds that might serve as substrates for energy yielding reactions within the erythrocyte. Particular attention was paid to glucose (3) and purine nucleosides (4, 7) which have been shown to prolong the viability of stored erythrocytes and to retard their progressive lysis and diminished resistance to hypotonic solutions. It may be noted, however, that the effectiveness of the purine nucleosides in the preservation of erythrocytes has recently been questioned (8).

Reduced nicotinamide adenine dinucleotide and the reduction of oxidized glutathione in human erythrocytes

The ability of reduced nicotinamide adenine dinucleotide (NADH), generated through the activity of lactic acid dehydrogenase, to support the reduction of endogenous oxidized glutathione in intact human erythrocytes and in hemolysates was investigated. Rapid initial oxidation of endogenous reduced glutathione was effected with methyl phenylazoformate. Freshly obtained normal erythrocytes and erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were unable to regenerate reduced glutathione upon incubation with lactate. Only normal erythrocytes were capable of reducing oxidized glutathione after preincubation with glucose, inosine, or a medium which promoted the synthesis of increased amounts of intracellular NAD. This regeneration of reduced glutathione could be explained by the generation of reduced nicotinamide adenine dinucleotide phosphate through the metabolism of accumulated phosphorylated intermediates of glycolysis. Hemolysates prepared from both normal erythrocytes and from erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were able to reduce oxidized glutathione in the presence of added lactate and NAD. The results obtained indicated either an inability of the intact erythrocyte to utilize the NAD at the concentrations attained or an altered behavior of the system for the regeneration of reduced glutathione after lysis of the cell.

THE INCORPORATION OF NICOTINIC ACID AND OF NICOTINAMIDE INTO THE PYRIDINE NUCLEOTIDES OF ERYTHROCYTES AND RETICULOCYTES OF RABBITS IN VITRO

There is abundant evidence that loss of intracellular structure and alteration in metabolic activity occur as mammalian reticulocytes mature to become nonreticulated erythrocytes (2). Loss of the ability to synthesize heme (3), proteins (3, 4), lipids (5-7), purine nucleotides (8), and pyrimidine nucleotides (9) from small molecule precursors accompanies the disappearance of mitochondria, ribosomes, and ribonucleic acid from reticulocytes. An intact Krebs tricarboxylic acid cycle and a complete cytochrome system are not present in the mature mammalian erythrocyte (10). The concentration of ATP is approximately 2.5 times greater in rabbit reticulocytes than in the mature erythrocytes of rabbits (11). Further changes, including a progressive decrease in the activity of several enzymes of the glycolytic pathway (12) and of the hexose monophosphate shunt pathway (12, 13) and in the concentration of ATP (12, 14), are associated with aging of mature mammalian erythrocytes in vivo. The concentrations of DPNand of TPN of avian and human erythrocytes were reported to decrease during storage in vitro (15), and the concentration of DPN in human erythrocytes was found to