Eugene J. Krob

Alterations in membrane surface properties of reticulocytes during maturation as determined by partition in two-polymer aqueous phase systems

Abstract Changes were studied in the partition in aqueous dextran-polyethylene glycol two-phase systems of normal rat reticulocytes as a function of maturation. Diminution of partition of erythrocytes with increasing age has previously been described [10, 11, 17]. Together these data provide a complete picture of changes, as measured by partition, in membrane surface properties of red cells in the circulation during maturation and aging. 1. 1. Young rat reticulocytes have the lowest partition in two-polymer aqueous phases of any red cells in the peripheral circulation. These cells were radioactively labeled in vivo, isolated by countercurrent distribution and transfused to normal rats. The recipients were bled periodically and their blood, in turn, was subjected to countercurrent distribution. It was found that the partition of reticulocytes increases as they age until it becomes the highest partition of any red cells in the circulation. This process, in the rat, takes about 24 h. 2. 2. On aging, the partition of erythrocytes gradually diminishes [17] until the oldest erythrocytes have a partition close to that of the youngest reticulocytes. This process takes the entire life-span of the red cell (about 55 days in the rat). Surface charge has been implicated as being a major determinant of erythrocyte partition [7,18,19] and it is not unlikely that the increase in partition that accompanies reticulocyte aging reflects an increase in its surface charge. 3. 3. There is a correlation between the partition of young reticulocytes and those cells which incorporate 14C-valine into hemoglobin in vitro. 4. 4. The changes in the cell membrane that result in altered partitions during reticulocyte and erythrocyte aging could not be duplicated in in vitro incubation of labeled rat red cells. 5. 5. It is shown, by comparison with untreated rat red cells, that the contact with the polymers in the aqueous phase systems used as well as the mechanical aspects involved in countercurrent distribution have no apparent effect on the survival of the red cells when transfused to normal rats. The protective effects of the phases on cells have previously been discussed [16].

Partition and countercurrent distribution of erythrocytes and leukocytes from different species

Abstract 1. 1. The partition of red blood cells, white blood cells and platelets from a number of different species in aqueous, dextran-polyethylene glycol 2-phase systems was studied. The partitition coefficients of these cells were found to be species dependent; they provide, in some cases, a method for the separation of leukocytes from erythrocytes by countercurrent distribution. 2. 2. The separability of red blood cells from white blood cells by countercurrent distribution is illustrated for rabbit and for rat. The partitition coefficients of their red and white cells are sufficiently different that a separation is feasible. Distribution curves of erythrocytes and leukocytes from these two species are presented as confirmation of the partition studies. Since red blood cells of different ages are separated by countercurrent distribution [11], it follows that by appropriate choice of phase system and species one can obtain red blood cells of different ages free of white blood cells in one operation. 3. 3. It is also shown that the sequence of partition coefficients for erythrocytes and platelets from a number of species is similar. This may be fortuitous or indicate a similarity of those surface properties which determine the partition of cells in these phase systems. 4. 4. Countercurrent distribution of white blood cells from lamb (and rabbit but not human) results in a 2-peak curve. Preliminary examination of the lamb cells in the different cavities of the extraction train indicates the presence of both granulocytes and lymphocytes under both curves. It is possible (although by no means established) that we have resolved two populations of granulocytes and two populations of lymphocytes. Among other possibilities is that intact and damaged leukocytes have been separated. Studies with white cells are being continued. 5. 5. A few remarks on phase systems and the possible relation between these and the cell characteristics which determine the partition of cells are included.

Factors in the partition of red blood cells in aqueous dextran-polyethylene glycol two-phase systems

Abstract The usefulness of countercurrent distribution of cells in aqueous, buffered two-polymer phase systems is illustrated by the segregation of erythrocytes of different ages, the resolution of two populations of reticulocytes, the separability of red blood cells from white blood cells, a study of membrane characteristics of red cells from different species 1,2 . We now report the effects on erythrocyte partition of a number of factors ( e.g. I , pH, temperature, hypertonicity or hypotonicity, ABO blood group, washing of cells and anticoagulant). It is also shown how some of these parameters can be used to obtain desired partitions for (red) cells in a given investigation. In the dextran-polyethylene glycol phase systems that we have investigated, an increase in I (with constant isotonicity) appears not to alter the partition of red cells except with phosphate in which a decrease in the partition coefficient is observed. Increase in pH or temperature causes an increased partition. Different degrees of hypotonicity give similar partitions which are somewhat greater than those found in isotonic phase. Increasing hypertonicity leads to a rapid decrease in the partition coefficients. Human red cells are found to have the same partition independent of the ABO blood group to which they belong. Washing of cells or anticoagulant used (acid-citrate-dextrose, heparin, EDTA) has no effect on their partition. In view of the previously established correlation between electrophoretic mobility of red cells and their partition coefficients 3 we point out, in the course of the discussion, those instances in which the electrophoretic mobility and partition of cells are similarly affected by changing a given parameter, and those instances in which the effects are markedly different. In the latter instances partition of cells in aqueous two-polymer phase systems and electrophoretic mobility appear to measure different membrane characteristics.

Separation of lymphocytes and polymorphonuclear leukocytes by countercurrent distribution in aqueous two-polymer phase systems

The littoral cell population, on the other hand, is part of the RES; there is evidence that extensive transformation and migration of RES cells occurred within the liver during regeneration, and up to 50 % of the RES cell population in the liver remnant, particularly during the early period of regeneration, migrated to the liver from extrahepatic RES reserves to augment the deficiency [l, 21. The size of the proliferating pool, therefore, would depend, in part, on the contribution of the hepatic RES, and, in part, on the extent of cellular mobilization and migration from extrahepatic sources. The relative proportions could be expected to change rapidly with time, particularly as the physiological stress placed on the liver progressively decreases during regeneration. In the present studies, the actively proliferating pool of parenchymal cells was 85 %. The actively proliferating littoral cell population was 3 %; the potentially proliferative pool was >50 %, but this value could not be measured with precision.

Hydrophobic affinity partition in aqueous two-phase systems of erythrocytes from different species. Systems containing polyethylene glycol-palmitate.

Abstract We have examined the partition of erythrocytes from different species in dextran-polyethylene glycol aqueous two-phase systems containing sodium chloride and small quantities of polyethylene glycol-palmitate. The partition of erythrocytes in these phases depends on the hydrophobic affinity of the membrane surface to the palmitoyl residue. It was shown that: 1. 1. The partition coefficient of human erythrocytes is not decreased by neuraminidase treatment indicating that membrane charge of cells is not a determinant of their partition. 2. 2. The partition of erythrocytes is species-specific and appears to depend, to a great extent, on the ratio of poly/mono-unsaturated fatty acids in their membranes. 3. 3. By combining isotopic labeling techniques with countercurrent distribution of rat red cells age-dependent alterations in the partition coefficient of erythrocytes were found which ( a ) reflect changes in the membrane lipid composition of red cells during maturation and aging; and ( b ) are different from the changes observed in a dextran-polyethylene glycol two-phase system with phosphate but no polyethylene glycol-palmitate (a system which reflects membrane charge [3]). 4. 4. Changes in partition coefficient of human erythrocytes as a function of storage occur.

Surface alterations of erythrocytes with cell age: rat red cell is not a model for human red cell.

Summary The charge-related surface properties of human and rat young and old erythrocytes were examined by partitioning in an aqueous dextran-poly(ethylene glycol) phase system having an electrostatic potential difference between the phases and by electrophoresis. It was found (a) that while rat red cells undergo changes in surface charge-related properties as a function of cell age which are detectable by partitioning, human erythrocytes do not; (b) that analytical particle electrophoresis after countercurrent distribution confirms both the alteration in rat and its absence in human erythrocytes; and (c) that particle electrophoresis cannot detect the age-related charge-associated alteration in rat red cells without prior cell partitioning.

Partition of rat liver cells in aqueous dextran-polyethylene glycol phase systems☆

Abstract Enzyme treatments change the surface charge of cells and thereby affect their partition in two-polymer aqueous phase systems. A mechanical dispersion method was therefore used for the preparation of normal and regenerating rat liver cells although changes in the surface properties of the cells so obtained may also occur. 1. 1. Dextran-polyethylene glycol phase system. The partition coefficient and electrophoretic mobility of regenerating liver cells is higher than that of normal liver cells. Countercurrent distribution of liver cells gives rise to two peaks: the left being larger than the right one for normal cells and the reverse holding for regenerating cells. Liver cells show a correlation between increasing electrophoretic mobilities and increasing partition coefficients through the bulk of the distribution curve. Incubation of liver cells (with 14C-valine) leads to distribution curves that are frequently shifted to the left and distorted but indications are that some separation of differently labeled cells occurs. 2. 2. Dextran-polyethylene glycol phase systems also containing a positively charged polymer. Countercurrent distribution of normal and regenerating liver cells results in single-peak curves. Liver cells from rats injected with india ink particles show a separation of hepatocytes from cells containing india ink particles (histiocytes). The latter are at the extreme right end of the distribution but have lower electrophoretic mobilities than the hepatocytes. Thus the histiocytes probably have a lower electrokinetic charge density than the hepatocytes but a higher charge density in the total peripheral zone (reflected by their partition). Mono- and bi-nucleated liver cells also appear to have different surface properties since their partial separation is effected by countercurrent distribution. Incubation of liver cells (with 14C-valine) does not affect their distribution curves; the more highly radioactively labeled cells are found under the right and left ends.

Effect of acetaldehyde and glutaraldehyde fixation on the surface properties of red blood cells as determined by partition in aqueous phases.

Abstract A correlation between partition of cells in dextran/polyethylene glycol aqueous phase systems and their relative electrophoretic mobilities is often in evidence. Absence of such a correlation is indicative that partition measures surface properties other than charge at the plane of shear. Acetaldehyde- and glutaraldehyde-fixed erythrocytes were partitioned in two-polymer phases and their electrophoretic mobility examined in order to investigate the circumstances under which the above-indicated correlation holds. Aspects of the effect of fixation of cells on their surface properties was thereby obtained. 1. 1. Acetaldehyde-fixed and normal human red blood cells have similar partitions and similar electrophoretic mobilities; glutaraldehyde-fixed red cells display markedly increased partitions and mobilities. 2. 2. Lipid-extracted aldehyde-fixed cells have substantially increased partitions, but unchanged mobilities, when compared with the fixed cells from which they were prepared. This indicates that the removal of lipid exposes a higher density of charge groups deeper in the membrane than is measurable by electrophoresis under the conditions used. 3. 3. The countercurrent distribution obtained with fixed cells depends on the length of time chosen for phase settling. Short times result in a single distribution while longer settling times give rise to a two-peak distribution. The latter phenomenon probably arises from a time-dependent aggregation of the fixed cells in the phases. 4. 4. Electrophoretic examination of the glutaraldehyde-fixed cells from different cavities along the extraction train indicates that the fixation process does not eliminate differences in the relative electrophoretic mobilities of erythrocytes of different ages.

Membrane surface properties of reticulocytes from rats rendered severely anemic with phenylhydrazine as determined by partition in aqueous phase systems

Abstract Partition was studied in aqueous dextran-polyethylene glycol two-phase systems of reticulocytes from rats rendered severely anemic with phenylhydrazine. 1. 1. The countercurrent distribution of reticulocyte populations from phenylhydrazine-injected rats indicate that these are highly heterogeneous. The distribution patterns obtained include separation of such reticulocytes into two distinct sub-populations, a broad (and therefore obviously heterogeneous) curve, or a distribution intermediate between these. 2. 2. A part of the heterogeneity of the reticulocyte population is due to the degree of maturation of these cells. Highly reticulated reticulocytes (with the highest protein synthetic activity) have the lowest, lightly reticulated reticulocytes an intermediate and erythrocytes the highest partition. 3. 3. The partition of reticulocytes is lower than that of normal erythrocytes and is thus similar to the partition of the youngest reticulocytes from normal rats [6]. Reticulocytes from phenylhydrazine-injected rats are shown to maintain this (low) partition while they age. They differ thereby dramatically from normal rat reticulocytes the partition of which increases rapidly as they mature. 4. 4. Reticulocytes from phenylhydrazine-injected rats (‘abnormal’ reticulocytes) do mature to erythrocytes. But these, in turn, have a lower partition than erythrocytes from normal rats. Surface charge is a major determinant of cell partition in dextran-polyethylene glycol phases. The lower partition of reticulocytes from phenylhydrazine-injected rats and of the erythrocytes into which they mature therefore reflects a lower surface charge on these cells compared to their normal counterparts. 5. 5. A large percentage of the abnormal reticulocytes disappears rapidly from the circulation both in the phenylhydrazine-injected animal and when transfused to normal rats. This rapid disappearance may well be related to the abnormal membrane surface characteristics of these cells.

Surface differences between erythrocytes from arbitrarily chosen (presumably hematologically normal) individuals detected by cell partitioning.

Differences in membrane surface properties (both charge-associated and lipid-related) of erythrocytes from any two arbitrarily selected individuals can be detected by use of a purely physical method: cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems. The procedure consists of isotopically labeling (with [51Cr]-chromate) aliquots of red blood cell populations to be compared. Such labeled cells are mixed with an excess of unlabeled erythrocytes from the other individual and the mixtures subjected to countercurrent distribution in either a charge-sensitive or a non charge-sensitive aqueous phase system. As control we also prepare mixtures of labeled cells with unlabeled cells from the same individual to ascertain that the label per se has no influence on the cells partitioning behavior. The distribution curves are analyzed for total cells (in terms of hemoglobin absorbance) and labeled cells (in terms of counts/min). Changes in the relative specific activities through the distribution curves are routinely obtained when cells from different individuals are used and are indicative of subtle differences in surface properties of such erythrocyte populations.