Claude F. Reed

Phospholipid exchange between plasma and erythrocytes in man and the dog

The turnover of the four major erythrocyte phospholipids has been studied with (32)P, both in vivo and in vitro, in man and the dog. Phosphatidyl serine and phosphatidyl ethanolamine appeared to be stable erythrocyte lipids in both species. Turnover of the phosphate moiety of lecithin and sphingomyelin in the circulating erythrocytes of these two species seems entirely due to an exchange of the whole molecule with the corresponding plasma compound. Exchangeable and nonexchangeable pools of these two cellular lipids were found. In man about 60% of erythrocyte lecithin is exchangeable. The 12 hr fractional turnover of this pool is approximately 13%. Only 30% of the sphingomyelin in human cells appeared exchangeable; this portion had a 12 hr fractional turnover of about 14%. Similar results were obtained in the dog except that in this species about 75% of the erythrocyte sphingomyelin was exchangeable. Inorganic (32)P was not incorporated into any of the four major phospholipids in either species. The present findings aid in estimating quantitatively the effect of plasmaerythrocyte lipid exchange on red blood cell phospholipids.

Changes in the properties of human erythrocyte membrane protein after solubilization by butanol extraction

1. 1.|Human erythrocyte membrane protein is readily solubilized and freed of most of the membrane lipid after a single extraction of an aqueous suspension of hemoglobin-free ghosts with n-butanol. The technique yields a mean recovery of 83.1% ± 3.8 (S.D., n = 10) of the membrane protein in the butanol-saturated water phase. 2. 2.|The mean amount of lipid present in the water phase after a single extraction is 4.8% ± 3.9 (S.D., n = 6) of the weight of protein. Loss of lipid is symmetrical except that a relatively high proportion of phosphatidyl serine remains. Analysis of the chemical composition of the protein indicates that it is a glycoprotein, which contains hexose, hexosamine, fucose and sialic acid. Carbohydrates account for about 9% of the protein by weight. 3. 3.|Approximately 98% of the protein has a single electrophoretic mobility and appears as a single peak in the void volume after gel column chromatography. Ultracentrifugal analysis, however, demonstrates heterogeneity of the protein. The protein is soluble at neutral pH in salt-containing aqueous media and has an isoelectric point between pH 4.0 and 5.0. 4. 4.|Solubilization of membrane protein by treatment with n-butanol results in a minimal (5%) increase in p-chloromercuriphenylsulfonate titratable SH− groups. 5. 5.|The cation-independent nucleosidetriphosphatase and alkaline monoester phosphydrolase activities of intact stroma were recovered to the extent of 73% to 107% in the solubilized protein. Recoveries of acid phosphohydrolase were 40% at pH 7.0 and 30% at pH 6.0. 6. 6.|The prominent cation activation of both nucleosidetriphosphatase and acid phosphohydrolase activities in stroma was virtually lost on solubilization of the protein. In the case of nucleosidetriphosphatase the concentration of butanol necessary for loss of activation by Mg2+ or by Mg2+, Na+ and K+ was sufficiently high (above 500 mM) to cause disintegration of structure.

Erythrocyte lipid loss in hereditary spherocytosis.

Probable manifestations of the intrinsic corpuscular defect leading to shortened red blood cell (RBC) survival in hereditary spherocytosis (HS) have been induced by in vitro incubation of these erythrocytes (2-4). The classical in vitro changes that have been described are a marked increase in autohemolysis in the absence of metabolized substrate and an increase in osmotic fragility. Young, Izzo, Altman, and Swisher (4) showed that a number of substrates utilized by RBC to produce ATP could substitute for glucose in preventing the exaggerated in vitro hemolysis in HS erythrocytes. The exact role of cellular ATP in preventing these changes has, however, been debated. Selwyn and Dacie (3) suggested that, particularly in the absence of metabolized substrate, a premature degeneration of the membrane occurred in HS erythrocytes during in vitro incubations. In a preliminary report (1), we described an in vitro loss of lipid in HS erythrocytes not found in normal cells that was minimized, but not prevented completely, by the presence of glucuose. These findings appeared to be a specific manifestation of the membrane degeneration postulated by Selwyn and Dacie (3) and to represent an example of the mechanism, originally proposed by London and Schwartz (5), whereby RBCcellular energy is utilized in preventing changes in membrane lipids; in HS erythrocytes, it appeared that utilization of energy through this pathway was inefficient.

The role of potassium in the prevention of alkalosis

Abstract 1.1. Loads of sodium bicarbonate were administered to normal rats in drinking water along with known amounts of potassium and chloride. Alkalosis did not develop after two weeks as long as potassium chloride intake exceeded 0.5 mM/kg./day even though sodium bicarbonate intake amounted to 10 to 30 mM/ kg./day. It was not possible to produce alkalosis in this way in the absence of potassium deficiency, as demonstrated by muscle analysis. 2.2. Sodium bicarbonate, 15 mM per kg., was injected into normal and potassium-deficient rats, and urine was collected for the subsequent twelve hours. Serum and muscle analyses were obtained at the end of that time also. Normal animals excreted 10 mEq. of sodium and 5 mEq. of potassium per kg. of body weight in the twelve-hour period following loading. Essentially no chloride was excreted and alkalosis was not present after twelve hours. Potassiumdeficient animals excreted only 6 mEq. of sodium per kg. and no potassium. Almost 1 mEq. of chloride per kg. of body weight was excreted by the potassium-deficient animals and severe hypochloremic alkalosis was present twelve hours after loading. 3.3. Potassium bicarbonate, 3 mM per kg., was injected twice a day into potassium-deficient rats with hypochloremic alkalosis. No sodium or chloride was administered. After 24 mM per kg. of potassium bicarbonate had been administered, serum bicarbonate concentration fell to normal and serum chloride concentration rose to normal. This correction was associated with relatively little increase in bicarbonate excretion and with essentially no change in urinary pH or titratable acidity. Cation was excreted with organic anion, a large part of which was shown to be citrate.These results suggest that citrate may be substituted for chloride in the renal defense against alkalosis so that sodium is excreted without chloride and without change in urinary pH. Potassium would seem to be essential for the maximal conservation of chloride in this way. 4.4. These findings suggest that potassium deficiency may alter the ratio in which sodium and chloride are reabsorbed from glomerular filtrate. Such an alteration leads to changes not only in extracellular concentrations but also in extracellular volume.

An inherited molecular lesion of erythrocyte pyruvate kinase: Identification of a kinetically aberrant isozyme associated with premature hemolysis

Atypical cases of heritable hemolytic anemia have been noted that conform clinically and biochemically to anemias of the pyruvatekinase (PK)-deficient type, except for the presence of apparently adequate quantities of erythrocyte-PK activity by the usual assay procedure. Investigations of four such anomalous cases, occurring in two unrelated families, are presented. Erythrocytes contained a kinetically aberrant isozyme of pyruvate kinase (PK(2)). Michaelis constants for the pathologic isozyme relative to phosphoenolpyruvate were over 10-fold greater than control values, but no kinetic abnormality was evident for the second substrate, adenosine diphosphate. PK(2) exhibited a pH optimum almost 1 U lower than the wild enzyme form (PK(1)). Significant differences were also evident in the functional stabilities of the isozymes. Leukocytes were unaffected. Family studies revealed paternal heterozygosity for quantitative PK deficiency of the usual type. Clinically normal maternal relatives and some siblings demonstrated intermediate deviations in erythrocyte-PK kinetics and reaction characteristics compatible with coexistence of normal PK(1) and kinetically abnormal PK(2). Hemolytic anemia in the propositi appeared to require simultaneous inheritance of the gene governing PK(2) production and its presumed allele resulting in quantitative PK deficiency. Both genetic defects were traced through three generations, the defective gene in both instances apparently resident on autosomes.A revision of the PK assay technique is suggested, since catalytic inefficiency of PK(2) was manifested only at low substrate concentrations and was therefore undetectable at the relatively high phosphoenolpyruvate levels employed in the conventional assay.

A radioactive label for the erythrocyte membrane.

Abstract A new radioactive reagent, diazotized [ 131 I] diiodosulfanilic acid, which binds specifically to protein groups of the erythrocyte membrane, is described. The labeling reaction and some of the effects of varying amounts of the bound isotope on red cell structure and function have been described. Among the advantages of this membrane label over others available are the following:(1) The diazonium salt of the diiodocompound is less soluble than that of sulfanilic acid and can therefore be precipitated from the diazotization mixture, washed, and rendered free of reactants and impurities. (2) Effective membrane labeling can be achieved with relatively little damage to the cell. (3) It can be prepared by a γ-emitting radioisotope at very high specific activity. (4) Preliminary data suggest that it may be utilized to study the fate of the red cell membrane in vivo .

Incorporation of orthophosphate-32P into erythrocyte phospholipids in normal subjects and in patients with hereditary spherocytosis

The in vitro incorporation of inorganic (32)P into erythrocyte phospholipids has been studied in normal subjects and in splenectomized patients with hereditary spherocytosis (HS). Phosphatidic acid (PA) was the only lipid measurably labeled in both kinds of cells. The actual turnover rate of PA phosphate was determined by simultaneously isolating inorganic phosphate (P(i)) and adenosine triphosphate (ATP) and determining their specific activities. This turnover is very small: 1.3 mumoles P/liter of erythrocytes per hr in normal cells and 4.0 mumoles P in HS erythrocytes when either ATP or cellular P(i) is considered the immediate precursor. This value represents less than 0.1% of the total membrane lipid phosphate. Incorporation of added (32)P(i) into the other phosphatides, including phosphatidyl serine, was essentially zero in both kinds of cells. The effects of stimulation and inhibition of active cation transport, metabolic depletion, and extracellular phosphate concentration on both the degree of labeling and the actual turnover of PA phosphate were studied. In any given experiment, the degree of labeling of PA depended on the specific activities of the other intracellular phosphates (P(i) and ATP). The actual turnover rate of PA phosphate, however, did not vary with active transport or metabolic depletion. The greater turnover of PA phosphate in HS erythrocytes may be due to the somewhat younger age of these cells. The results suggest that the very low turnover of PA phosphate in erythrocytes is mediated by nonspecific enzyme reactions, and that it is quantitatively insignificant in both normal and HS erythrocytes. The results also emphasize the importance of measuring intracellular phosphate precursors in any study evaluating cellular phospholipid turnover from added (32)P(i).

Erythrocyte Energy Metabolism in Hereditary Spherocytosis

The incorporation of extracellular orthophosphate-(32)P into cellular ATP, 2,3-diphosphoglyceric acid, and inorganic phosphate has been measured over a period of 6 hours in vitro in red blood cells from normal subjects and from patients with hereditary spherocytosis who had undergone splenectomy. The pattern of labeling of the intracellular compounds was found to be the same in both types of red blood cells, as reported by other workers using much shorter periods of incubation. In addition, in the present study it was possible to compare the net flux of extracellular phosphate into ATP between the two groups of erythrocytes. These latter results suggest that the actual turnover rate of ATP was not abnormal in these patients with hereditary spherocytosis.