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From www.bloodjournal.org by guest on August 9, 2017. For personal use only. Red Cell Aging. I. Surface Charge Density Sialic A cid Content of Density-fractionated Human Erythrocytes By G. It has red been cell V. F. Seaman, suggested that surface-charge R. J. Knox, decreases density F. J. Nordt, in ORMAL 120 days cells are primarily istics HUMAN and the RED degree CELLS circulate of random cell eliminated by phagocytic in the spleen, but also of the old red cell cells in the which lead D. H. Regan charge density for the extreme 5% fractions based on the electrophoretic behavior of the cells. However, slightly lower levels of sialic acid were consistently observed in the densest red cell subpopulations. These observations and other cited evidence are consistent with the view that during its life span in vivo, portions of the red cell membrane are lost as the result of innumerable cell-cell and cell-vessel wall contacts during the passage of the cells through the circulatory system. Such losses would account for decreases in the level of membrane constituents per cell, such as sialic acid, but would not require that the concentration of those constituents be altered in the remaining membrane. Thus such features as surface-charge density could remain unchanged even though the total sialic acid content per cell was reduced. accompany aging in vivo and reflect alterations in the cell surface which play a critical role in the recognition and elimination of effete erythrocytes by macrophages in the reticuloendothelial system. The bases for this suggestion are reports that the surfacecharge density progressively decreases for red cell subpopulations sampled from regions of increasing density within the whole population where the least dense fractions are enriched in young erythrocytes and the most dense in old erythrocytes. We have attempted to reproduce these results and have examined the relationship between cell surface sialic acid and cell surface-charge density for the extreme density fractions of fresh human red cells. Contrary to the earlier reports, we observed no differences in net surface- N and and in the bloodstream destruction is low.’ of the reticuloendothelial liver and bone marrow.2 to its disposal are not for 110Senescent system (RES) The character- understood but are thought to involve changes in one or more physicochemical properties of the cell. Both deformability3 and the net surface charge4 of the cells are properties postulated as possible initial key factors in the recognition and elimination process. would Progressive decreases in cellular deformability during impair or slow its passage through the microcirculation. erythrocyte has become microcirculation hypothesis does the recognition sufficiently rigid and is no longer aging of the cell Once the aged able to traverse the of the spleen, sequestration occurs. While the deformability account for shortened red cell survival in some disease states, and eventual phagocytosis of the normally aging cells remains unexplained. From the Department Submitted April Supported Address Health © Blood, by USPHS for reprint Sciences /977 of Neurology. 4, /977: by Grune accepted Grant requests: Center. 3/8/ & Stratton. Vol. 50, No. 6 (December), HL G. University July /8284 1977 iSSN of Oregon Health National Heart. Sciences Center. Portland. Ore. /977. from the V. F. Seaman, 5. W. Sam inc. /2, Jackson Department Park Road, Lung of Portland, and Neurology. Ore. Blood institute. University of Oregon 9720/. 0006-497/. 1001 From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1002SEAMAN El AL. Another hypothesis charge density the altered progressively basis that densities.5’6 have cells implied is due groups to contribute and specific the The by sialic acid by the whose of the red then its poly- increasing the rapid on upon partial leads perhaps red carboxyl cell.9’0 been receiving and Morell” and de- to sufficient recognition sialic acid life span of the by autologous to the report macrophages normal we present a small Erythrocyte their loss of phagocytosis from mammalian the cells in vivo’2 in vitro.’5 recognition and The elimination erythI and significance mechanisms in either consistently was drawn citrate within method 16 x but blood trisodium tionated studies of the relationships between sialic acid level in density-fractionated between the mobility of “young” lower and level of sialic MATERIALS AND METHODS venipuncture from healthy the electropho- human red cells. We “old” red cells, but do acid in old cells. Fractionation Human a period 100 mm disodium of 4-6 Cell and Analytical suspended Particle Suspending NaCI-0.22 media gradients volts/cm Neuraminidase Vibrio cholerae M for different ionic strengths the pH adjusted to 7.2 were measured for 1 hr NaCI, or the at 27,000 approximately were washed and high-speed M listed g at the three 0.01 the analyses with water currents yr) into It was frac- centrifuga- top times and -30C in and 5,, bottom in 20-30 potassium spun a Sor- volumes phosphate of buffer, below. 0.2 used, by namely, NaHCO3 chamber M NaCI, and in in the lower ionic bath at 25.0 ± 0.15 M NaCI of equivalent apparatus and 0.03 ionic equipped M strength. with Ag a cylindrical chamber equipped strength media. Chambers were 0.lC and were operated at voltage <3 mA. of Cells and Sialic A cid A ssay N-acetylneuraminic neuraminidase were ± in a cylindrical suspended in 0.15 for cells suspended Treatment Membrane-bound medium 25 44 rophoresis in a constant-temperature of 5 0.144 (aged 1.5 mg Na2EDTA.2H2O/ml, to red cells with mobilities thermostated (PBS), donors method6”6 with aliquots) of two electrodes18 for cells platinum electrodes19 ester corresponding the in this Elect M sorbitol Electrophoretic fractions fractionation, saline phthabate (Il-mI adult tetraacetate(Na2EDTA.2H,O). anticoagulated tubes 7.4 phosphate-buffered 286 mOsm/kg, the blood polypropylene Following ethylenediamine hr by using centrifuge.’7 collected. by or of Murphy,’7 RC-2B with and of glycoproteins to of the of senescent glycoproteins cells; leads particles7 acid, charge plasma removal of membrane-bound neuraminidase decreases mobility and no differences observe AgCI with surface observation separated on density survival has of Ashwell recognize (age)-fractionated iron sialic if desialylation erythrocyte observations In this pH circulating the surfaceto is unclear. retic find were net reticuloendothelial phagocytosis of these vaIl decreased tuned of density colloidal membrane-bound holds aged are surface-charge of the of have RES RES. increases in of majority hepatic in the Enzymatic rocytes with vivo with acid in erythrocyte in part by the work hypothesis cells the interaction cells diminished loss elimination sialylation. removal the of on the red a partial senescent hypothesis is founded on charge in the old erythrocytes Studies that The role of sialic attention, stimulated the macrophages neuraminidase-treated lysine8 tion that the surface charge. The decreasing surface of their and/or provides and acid (VCN, (NANA) was N-acetylneuraminate released from the glycohydrolase, cells by EC treatment 3.2.1.18). From www.bloodjournal.org by guest on August 9, 2017. For personal use only. RED CELL AGING Typically, - 2 Behringwerke I0 x cells/mi suspension pared -20 37’C. and and and was NANA, A grade, calculated U/mI were acid was in 0.05 were (TBA),2#{176}alkaline Total NANA released Total number of RBC f the is packing suspension, the final included volume extraction. a final fluid saturated the was pre- cell count NaCl-0.0I acid cell I hr at tempera- was assayed methods. per M for g at room Sialic released Standard by VCN was then fluid Assay extracted used prepared by of the hypotonic cells l0 liters/mole-cm volume by w/v packed the VE the in the with TCA. times of at each the cell volume volume of of VCN the red added, and assay a 70% The mixture decantation of at room fluid NANA solution was cooled by ether of in water in TCA an ice fluid. 2 volumes which the digests VCN followed supernatant with from in (TCA) w/v the temperature supernatant of acid of final bath for The super- diethyl dilution ether factors step. in of were NADH. One the 2 oxoglutarate age of cells in PBS by in the per Henry cell at of NADH x diluted Red GOT cell lysates in approximately to Units 25’C; l0 fractions.22 et al.23 centrifugation mm to 4.82 aminotransferase, red International oxidation corresponds of by followed substrate for IU vivo expressed of nm 340 in as modified buffer,23 I ,zmole at L-aspartate: the volume assay Activities absorbance of of Karmen of a known phosphate for (GOT, marker method sonication conversion decrease the trichloroacetic mixed transaminase remained. the the with and three as an enzyme measured umes catalyzed Hct substances was 9- l0, weight recorded were red VE) + Methods was was Hct] V5 suspension, fluids centrifugation cell glutamic-oxaboacetic activity be 0.99, cell interfering sample of The _f. V supernatant) to red supernatant by water. was 2.6.1.1) (IU), they 10 vol- establish that where were calculated to NAD using Karmen units unit from 6.2 = for no I the x assay of 3 ml employed. Reticubocyte were measured computer Hycel counts were by electronic Analysis reagents Package and tubes was assumed. All reagents tilled twice grade reagent and estimated particle Red Data). according following ware Hemoglobin to their stored were in glass was assayed instructions. at distributions solutions and new methylene blue method.24 with an Electrozone-Celboscope-PDP centrifugation cell density standard in pyrex by the counting (Particle standards in microhematocrit 0.99 the of concentration with General intact final was fluid. presence followed computed Red the 7.4, (RBC/mI)V of stock supernatant 15 mm, EC M 1000 - supernatant)(V[l (NANA/mI assumed volume of The give were pH incubated resorcinol21 NANA containing of a known and at (RBC/ml) ofsupernatant on treatment natant PBS buffer-MIS NANA. and Total with mixed 10 mm Ehrbich,20 (NANA/mb fraction V the Experiments about was of liberated Calbiochem. in units/mi from where to cells in PBS, acetate it for analysis - vs sodium at 37’C; centrifuged for - cell of 100 Behringwerke - of erythrocytes M suspension from a suspension 1.5 ml of the suspension - VCN cell sampled obtained to of suspension of samples fluids added A stock aliquot to this incubation, thiobarbituric was concentration of 7.2. An added supernatant the a pH 0.3 ml ofSOO 5.6, Following ture solution an enzyme hematocrit. was weighed pH VCN to give volume at CaCl2, by 1003 were made up jugs. 15,000 g from The for cell volumes 5 mm. A by the phthalate analytical water fitted concentrations 8/M Mini- as cyanmethemoglobin Packed assessed Cell grade the with were packing ester measured fraction materials specifications in water for type water.25 RESULTS The hematologic in Table 1 and are in Table given parameters those for for the cell 2. In experiments the phthalate fractions 1 and obtained 2 the ester fractions by the anticoagulant of method,16 are presented Murphy method was 1 volume disII From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1004 SEAMAN Table 1. Hematologic Characteristics of Red Cell Fractions Obtained El AL. by the Phtha late Este r Method Exp. Population RBC No. Fraction Density* Retics. (%) MCH (pg) 1 lop#{243}% <1.085 2 lop 3 *Densiy 10% 5% GOT) (IU/RBC x lOfl) 5.5 31.5 96 32.8 1.2 34.6 100 34.6 >1.100 0.6 35.3 87 40.6 18.3 <1.085 6.7 37.2 104 35.8 20.8 16.9 - 1.9 35.6 97 36.7 19.8 11.8 20.1 - - - 1.2 35.7 86 41.5 19.6 lop7% <1.081 3.0 33.4 98 34.1 21.2 Whole - 1.2 30.3 88 34.4 16.9 7.5 31.5 79 39.9 16.8 4.9 Bottom 2% >1.097 0.6 listed is that of the phthalate ester N-acetylneuraminic glutamic-oxaloacetic w/v separation citrate technique counts, at 20’C. to 9 volumes whole tended and 13.5 acid. 2H2O/ml . mixture 6.6 transaminase. trisodium Na2EDTA locyte (fg/RBC) >1.103 GOT: mg NANAt (g/dl) Whole Bottom#{243}% tNANA: of 3.8% MCHC - Whole Bottom MCV (fi) cells of of blood; blood. to have the The higher bottom MCV, 5% in experiment cells of the GOT, top sialic characteristically 3 it was 5% acid, had 1 .5 with either and reticu- lower MCV, GOT, and reticulocyte counts, and consistently lower sialic acid levels. Electrophoretic mobilities for freshly drawn human erythrocytes and density fractions obtained by the phthalate ester method’6 and the method of Murphy’7 are presented in Table than 10% -of the whole strengths of 0.15 mean electrophoretic any of the fractions NANA was method,2#{176} and and 0.03 g mole/liter at 25#{176}C.No mobilities at a given ionic strength of whole populations analyzed. estimated in three ways: the resorcinol procedure.2’ experimentally and 3. The extreme density fractions, population, were examined by by adding to PBS in the Table 2. known absence of Hematologic red the which constituted electrophoresis at significant differences in were observed between TBA assay,2#{176}the alkaline Ehrlich The recovery of NANA was tested amounts of cells, incubating NANA to for a red 1 hr cell at suspension 37#{176}C,assaying Characteristics of Red Cell Fractions Obtained by the Method of Murphy Exp. Population No. Fraction Median Density Retics. (%) MCH (pg) MCV (fi) MCHC (g/dl) 5% 1.095 6.3 31.8 95 33.5 18.4 9.4 1.102 3.8 32.3 92 35.5 17.6 5.2 1.107 1.8 33.0 82 40.2 16.6 4.3 4 lop Whole Bottom 5 6 5% 1.095 6.6 27.4 87 31.5 17.8 10.7 1.103 1.2 27.2 80 34.0 16.0 3.9 Bottom 5% 1.104 0.5 27.6 77 35.8 15.9 2.0 1.098 6.3 30.4 89 34.2 17.8 9.7 1.104 2.1 33.1 87 38.0 17.3 6.1 1.107 0.4 33.2 79 42.0 16.2 4.8 lop 5% Bottom tGOl: GOTt (lU/RBC x 1011) 90% 5% Whole *NANA NANA* (fg/RBC) Middle lop 5% N-ocetylneuraminic glutamic-oxaloacetic less ionic acid. transaminase. From www.bloodjournal.org by guest on August 9, 2017. For personal use only. RED CELL Table AGING 3. 1005 Electrophoretic Mobilities Exp.#{176} No. Population Fraction 1 2 0.15 10% Whole lop 3 ± 0.10(46) (30) -1.72 ± 0.11 -1.10 ± 0.06 (30) -1.72 ± 0.09(46) (48) ± 0.05 -1.74 ± 0.09(12) -1.09 ± 0.07(60) -1.71 ± 0.09(50) 1.10 0.07(26) Bottom 7% -1.11 ± 0.05(32) -1.72 ± 0.09(28) -1.09 ± 0.05 (40) -1.76 ± 0.08(42) -1.09 ± 0.07 (20) -1.74 ± 0.06(20) 7% Bottom2% -1.12±0.08(20) Whole -1.10 ±0.04(24) -1.10 ± 0.07 (20) -1.82 ± 0.07 (20) -1.11 ± 0.07 (20) -1.84 ± 0.09 (20) 5% 5% 1.08 0.06(36) -1.79 ± 0.09(32) ± 0.04 -1.77 ± 0.10 (20) ± 0.06(28) -1.79 ± 0.08 (20) ± 0.06(28) -1.78 ± 0.07(20) 90% -1.08 Bottom 5% - 5% fractionation were coded by phthalate so that 1.10 (20) - 1.09 ± 0.07 (40) -1.74 ± 0.10(30) - 1.05 ± 0.08 (20) -1.72 ± 0.09(20) - 1.07 ± 0.08 (20) -1.70 ± 0.09 ester during -1.76±0.09(20) ± Middle 5% -1.73±0.12(20) -1.08 - 5% Bottom method; experiments the collection 4-6, of mobility data fractionation the ± for NANA, sample and then SD; the number of individual calculating the cells measured percentage appears recovery from an unfractionated VCN-modified cell suspensions treatment did plus not ether produce red cell population red cells with gave 0.1-0.4 extraction significant the TBA assay method These various experiments of the assay procedures fering observer was unaware of VCN. fg/RBC the decreases nor in parentheses. from data. No substantial differences in the amounts of NANA served for any of the three assay methods. Limited hemolysis not to interfere in the NANA assay system. Completeness acid (20) by Murphy identities. mobility treating treated (56) ± lop All samples -1.77 0.06 -1.70 Whole of the sample 0.05(10) ± 0.06(30) lop 1-3, ± -1.10 ± Whole *Experiments M NaCI -1.09 Bottom 6 0.03 4% lop 5 lity (iim/sec/V/cm)t Mobi M NaCl Bottom Whole 4 on the Basis of Density -1.09 - 3% lop iMean Electrophoretic . 6% Bottom Red Cells Fractionated Human Whole lop method. of Fresh by VCN the (-‘ was determined Supernatant fluids of TBA-positive supernatant in either fluids the level experimental recovered were ob2%) was shown of removal of sialic from of NANA by re- from such rematerial. TCA VCN diges.ts assayed by in the absorbance ratio (Abs 549 nm/Abs 532 nm). established no significant loss of NANA during any nor any influence on the assays of potentially inter- substances. DISCUSSION The need company for reliable A variety separation to describe the aging methods the biochemical and biophysical changes that red in vivo has impetus to the of human that of techniques on the basis separate cells red cell populations have been used, the most of differences in density. given on the basis may ac- search of their common involving centrifugal Stratification of erythrocytes age. From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1006 SEAMAN according to cell combined production with 59Fe labeling of the in rats by transfusion-induced populations age of rat has red been cells life span major would properties that during of the last ever, in these levels tions, of leukocytes which would enzyme, few days activity with contributions to erythrocyte, and Sass as with and reticulocytes influence the in GOT ported property decreases according of must be made Electrophoretic density.’8 The implies that brane at the electrophoretic charges per approximately sites could same the GOT The cells of the and can then any red cell’s the same cell age Statements which with for as life span. of indicator cell age of the compares in the cell age enzyme favorably low cell fracbasis for known, nor the transition denaturation due to other of levels characteristics separation with of the of methods re- any measured the life of the of are from of the depressed the hem atologic 2 indicate that the relationship near the end the The is not with age. Accordstudy. Howliterature, throughout some extent. associated fragmentation, Thus I and of cell in this reported red loss of occur fractions for cells cell cell, caution. net number of negative surface” data for human cell.34 If the limit be age, cell density be regarded mobility is recognized as a measure of net surface-charge electrophoretic mobility for red cells of different densities effective charges remains erythrocytes of sensitivity 5% or better, then approximately lost from an average red cell constant. per unit area that there are about of the electrophoretic 106 or without of mem- It is estimated somewhat apparent l0 from negative method fewer change is charge in the mobility. differences reported for distributed profiles to increasing activity “electrophoretic electrophoretic cells centrifugation, the end of cell age region ofthe studies were enzyme erythrocyte in the to in the literature. to the processes however, to subsehave been in vivo. If et al.22 have examined different levels for various enzyme activities and have most of the cofactor, pyridoxal phosphate.33 the red cell fractions given in Tables population to resort and MCV of aging fraction probably represent a considerwould tend to mask any changes in cell provides the most sensitive were used as indicators experiments, the relative reticulocyte values between least dense of red cell to obtain one the MCHC and MCV trends for the different cell 2) were in agreement with those previously reported concluded that GOT ingly, GOT activities decrease having during toward MCHC separated according to density.’7’3032 of a column of centrifuged erythrocytes the without AL. fractionation Suppression enables in both MCH a consequence of separation or MCV correlation from the ultracentrifugal in vivo.26’27 polycythemia ages from the most dense of older cells, which occur In this study (Tables I and MCH a range in a poorer While cells young cells, those able range in age by cells Reductions apparently as determinant changes of produce thereby resulting the older cells. red of different quent in vitro fractionation.28’29 reported in these studies, MCHC is the disproportionate demonstrated El in surface-charge by Danon and density Marikovsky5 ceptance in the hematologic literature, plausible mechanism for the elimination In the present study red cells from the between and “young” Yaari6 have and found “old” wide red ac- particularly as such results suggest a of the oldest cells from the circulation. extremes of the density distribution were From www.bloodjournal.org by guest on August 9, 2017. For personal use only. RED CELL AGING 1007 -:: 5% ,-) Fig. 1 . Mean electrophoretic mobility of human red cells as a function of density. ‘ Whole 6% o, Yaari’s Pop data; mean stippled boxes, range of mobilities for data in Table 1 ; vertical bars, ± SD calculated from Yaari’s data with the midpoint of each mobility interval and the quency for each interval a grouped data format. centages of the SD to percentage red Whole tion. ± refer total fre- using Per- cell popula- population derived point from 090 Yaari’s 1.095 1.100 examined. red cell The electrophoretic populations obtained summarized ences in Table for for red detected been 1.105 RED CELL data. the 3, showed extreme red cells from with ease, replotted the as (solid density is a monotonic no cell line) line). that and of The compared cell significant expected density, our 1.120 strengths procedures, mobility mobility distribution 1, in which with be seen from data suggest 1.115 (g/mI) at two ionic fractionation experimentally of the density seen from Fig. It can Yaari’s function made density populations. extremes may be (broken ionic strength ity versus cell measurements by the two 1110 DENSITY data differ- differences should Yaari’s own have data6 at our data mobilmobility indicate electrophoretic mobility is essentially invariant with age, at least over tral -95% of the red cell density distribution. The coefficients of variation for the sets of data in Table 3 average is probable that rather than The density any appreciable distribution indicate that the differences tected. The of presented of 12% ± 1.5 this variation dispersity for the data the separation in mobility data an average cells (19.4 much on the reported in Tables more fg/RBC originates from of mobilities cell fractions basis of cell density by Yaari, if real, 1 and 2 show that creases in sialic acid content resulting membrane with aging would tion of sialic acid. Consequently, on the electrophoretic charge groups, rather els of neuraminidase mobility, than the employed not from membrane young which total and measures number the lack the and been cells It 2) that de- contained than the old in contrast no differences be noted that fragmentation result in a change such losses of sialic cen- factors effective to have neuraminidase-susceptible NANA per cell versus 17.2 ± 1.4 fg/RBC), even though, to the findings of Danon and Marikovsky5 and Yaari,6 trophoretic mobility of the cells were observed. It should the the particle population. (Tables 1 and was ought the that 5.5%. instrumental in the examined been have physiologic the plot of electrophoretic that the electrophoretic whereas on in elecany deor loss of in the surface concentraacid would have no effect concentration of surface of charges per cell.’8 The high of significant additional release levof From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1008 SEAMAN Table 4. Total Cell Whole Bottom IBA 223 nmole/mI RBC 6.3 35 IBA 237 nmole/mI RBC 6.7 13 VCN Resorcinol 89 g/iO10 Acid Resorcinol lop 36 10 IBA-Resorcinol 135 .tg/mI RBC 12.2 Acid IBA 140 pg/mI RBC 12.7 14 - - 19.2 mg/100 ml RBC 17.4 37 - - 2.2 x g/ghost 22 38 39 - 10 14 348 ng/mg Hb 10.4 ng/mg Hb 11.6 Acid, NANAse IBA 327 ny/mg Hb 9.8 Acid IBA 1.72 omoIe/g Hb 16.0 Acid IBA 1.89 j.tmole/g Hb 17.5 10% Acid IBA 1.54 tmoIe/g Hb 14.3 Acid IBA 20.7 og/10’ RBC 20.7 14% Acid TBA 18.9 ug/109 RBC 18.9 309.3, MCH N-acetylneuraminic were packed NANA indicate susceptible 41 acid. made assuming a NANA molecular weight of of 30 pg. and 1.1 x 1010 perfringens upon that retreatment the enzymatic NANA should cells.’#{176} a check of literature NANA/cell) were the of whole cell populations release is essentially give a reliable estimate absolute values compiled (Table 4). obtained for under these conditions complete. Thus the VCNof total sialic acid content NANA/RBC, and converted, where possible, There was extreme variability arisen in one or originating from a combination the contribution fluids; (2) losses of sialic nate interfering substances; ing data used in calculating Schauer et al.35 comment neuraminidase-treated (adsorption with formic acid red of three ways: of interfering that cells ether for extraction removal of the of lipid When these measures were up to 200%, with indications based on aberrant absorption gion. of problems evident supernatant and ion resin with interfering not in the from same units the results have to elimisupportfluids exchange subsequent substances taken, they of interfering spectra in the published be- in NANA values in the supernatant during purification procedures intended or (3) erroneous assumptions or inaccurate or normalizing the results. TBA assay system. sults were elevated in the TBA assay the the in the values ranged 300%. blood cells may (I) elevation substances of sialic acid to an anion exchange acid) are both required to eliminate a result values into even reported for unfractionated red cell preparations, where tween 6.3 and 22 fg NANA/cell, a variation of more than The divergent results given in Table 4 for human red As 40 cells. Clostridium the (fg 11.7 386 tConversions ofthe As 9 1.17 pg x 102/RBC IBA *NANA RBC/ml 8.9 IBA 14% Bottom Acid RBC NANAset 10% Ref. No. Acid NANAse 11% Bottom NANA Acid, Whole lop Reported Calc.t NANA (fg/RBC) Acid, 20% lop NANA Assay by Acid VCN, Whole AL. Levels Reported for Human Red Cells and Subpopulations Obtained by Density Centrifugation NANA NANA* Release by Population El from clean-up elution in the report that chromophores 510-540-nm literature, studies rere- From www.bloodjournal.org by guest on August 9, 2017. For personal use only. RED CELL were AGING 1009 designed that would of our quantitation independent NANA TBA assays,2#{176}made it unlikely procedure in exactly suits by the different the values for establish the techniques for assay methods, that were accuracy, interfering the same way. methods under NANA validity, erythrocyte the alkaline Thus given not and reproducibility sialic acid. The use of Ehrlich,2#{176} resorcinol,2’ substances would influence the agreement obtained conditions is a strong influenced significantly three and each between indication by rethat interfering sub- stances. Our average value from whole human Cohen also et al.4#{176} and could find of 17.7 red cell ± 1.5 fg/RBC populations Greenwalt no and indicators of the chromophores interfering substances for the sialic acid released by VCN agrees well with those reported by Steane.4’ Like of interfering produced by TCA in and Greenwalt substances the TBA assay. ether treatments ence on the TBA results. The assayed NANA and resorcinol assays were within 20#{176},, of the and Attempts had no values TBA from assays. Canham32 estimated that the mean surface area and from the bottom l0#{176}fraction (phthalate ester method) tein, and lipid per cell. Cohen et distribution of the losses portionate of removal membranes major tions remaining are cells not to ions,45 charge and mechanical density between by the old of decrease of occur in chemmembrane pro- absence of However, if the for surface in partitioning toward of the membrane.46 young cells been the ratios behavior antisera,43 membrane of alterain two- binding permeability Differences have changes constituents, without dispro- even Evidence agglutinability macrophages,’5 and of properties altered. properties the basis et al.’ volume of red cells were about 10% less constituent. different differences systems,42 by autologous for some of the changes of cells has been shown of these variations the of Jancik major membrane fragmentation membrane have substantially is provided phase aqueous polymer lectins,44 phagocytosis cells the ratios of represent particular in old components in old any al.,4#{176} on and may any inilu- the alkaline Ehrlich We therefore con- cells from the top 20#{176}(, fraction. The magnitude is comparable to the extent of the changes that of the cells, such as decreases in total NANA, in the phospholipid suggested that most we examination to extract significant dude that the 2.8-fold difference between our results and those and Schauer et al.35 is not explained by interfering substances. than those for these parameters ical composition Steane,4’ by spectral of in surface- invoked to account in these properties. Now that the surface-charge density to be invariant under the conditions under which many measurements have must be sought. been made, other explanations for these age-related ACKNOWLEDGMENT The authors thank C. Tam blyn and B. Voyda for painstaking technical assistance. REFERENCES I. of the Red demic, Berlin red Blood NI. cell, Cell Berk in (ed PD: The Surgenor 2), vol biological DMacN 2. New (ed): York, life The Aca- 1975, pp 957 -1019 2. Beck WS: Iron metabolism chromic ogy. 27 anemias, Cambridge, the hypo- Beck WS MIT (ed): Hematol- Press, 1973, pp 50 3. Weed and in Mass, deformability. RI: The Am importance J Med 49: of erythrocyte 147 ISO, 1970 From www.bloodjournal.org by guest on August 9, 2017. For personal use only. SEAMAN 1010 4. Danon The D, Marikovsky sequestration erythroid nuclei, Structure and demic, 1971, 5. in D, #{233}lectrique de 127 1-1272, stained in (Paris) 253: blood cells field. of blood cells. 159, 1966 9. Cook acids blood surface the Katchalsky young Aminoff MA, of of erythrocyte. the Svennerholm acids. Acta Brody sialic in AG: the role hepatic circulating 41:99-128, The J, Schauer lifetime Z R: Sialic of rabbit transaminase, and Dacie ter. Adv Designation 26. Rigas Standard Physiol de- Chem cytes on 27. 355:395-400, 1974 Jancik fluence of aminic acid man. J, Schauer R, Streicher membrane-bound on the In- of Z Physiol erythrocytes Chem in 356:1329- Role 29. Durocher JR. of sialic 45:11-20, 15. 16. Mechanism Conrad survival. ME: L, by human Blood Murphy method Med JR: Y: of 64:668-674, Influence of centrifugation erythrocytes. red J Lab and Determinapopu- Med mechanism ship between Seaman in GVF: Electrokinetic Surgenor DMacN separation The R, J Clin 58: with re- Hillman Invest of a filtering 33. Van of RS: 50:1373 Dilla old Wasserman and PB: in J Lab iF: from in 69: bone geometry of explained by Res 25:39 H: Med 1967 erythrocytes Circ gravity Erythrocyte recovery Difference Walter LR: Relationa discon- Clin 2 13:708-709, human I, I. specific Spalding Nature as 1974 aging. during mechanism. Fischer G, cell age MA, Hemoglobin erythrocytes 29:277-279, gradient. Canham and red of ultracentrifugation distribution arrest. BIut NT: 5, Lurinsky cell by 1967 32. Red area density marrow 82:334-341, behavior (ed): Med Ultracentrifugal Oakes vivo. Tijmes 659-674, young cells, Clin 291, 1963 AFW, tinuous 31. 1973 18. Ultraerythro- erythrocytes projection Piomelli The volume temperature the in of cell age. evaluated 1964 of on Clin of Proc cell K: human M: human AM, aging indication 1975 Marikovsky distribution Clin removal macrophages. 72:3521-3525, D, density of Hjelm of Morselt content 30. Sci USA J Lab 17. MMB: Danon of lation. in erythrocyte cells Acad tion acid RC, 1975 Kay senescent NatI Payne Swisher of Wa- Materials 1378, 1971 133 I, 1975 14. RD. and basis of age. J Lab Ganzoni cell Reagent Testing spect to cell age. Blut9:284 Red HemaLiving- 1961 Garby 28. N-acetylneur- survival Hoppe-Seylers H-J: Practical Churchill for for glutamicic-pyruvic dehydrogenase. SM: Dl 193-74 DA, Koler the OJ, 1960 York, Society fractionation 13. acid fractionation 242- 246. of Specification American Golub glutam 1-398, Lewis New sur- acid-A N, spectrophotometric lactic JV, PW: red cell age. 1964 inase, 34:38 5). Spear of determination Pathol of erythrocytes. Biophys E, Chiamori the centrifugal estimation resorcinol-- indicator transam (ed sialomucoids. Biochim Revised for 24. of Quantitative 10:21-26, J Clin quantitative colorimetric an Ri, 5: tology the Vorsanger as stone, 1975, p 79 recognition 182:642-644, acid and their method. MD, activity Biol glycoproteins. DH, 1957 J 1974 of the L: acid 23. Henry Am to Heard microelectro- 1961 11. A ClinChimActa OV, acid for 24:604-611, 25. Morell of Hoppe-Seylers of D: Methods hydrolysates 21. oxaloacetic 1961 R, Nature to of sialic 1962 carbohydrates Jancik charge Academic, for particles. J 8 1:384-392, Berkman GVF: 19 1:44-47, Madoff G, transport Seaman contribution charge Ashwell DH, Flemans apparatus of N-acetylneuraminic Biochem old 124:154- electrokinetic 237:1992-2000, terminant red 20. estimation 22. Sass A: and Acta Nature EH, JL: The Enzymol of ofsmall Enzyme D, York, 1958 charge of Biophys Heard and surface An hydrochloric cells 1969 Danon erythrocyte. 10. Eylar and phoresis methods GMW, the human Oncley for polylysine Biochim micro- red AD, GVF: Blood Electron old 43:1-7, Y, by D: iron Biol Marikovsky 12. de Bangham Seaman #{233}rythrocytes [DI electric and colloidal J Cell Agglutination and an Y, Danon with face entre Sci of young evaluation. 11. Difference 2), vol 2. New (ed AL. 1135-1229 19. Cell Aca- 1969 analysis Chem Y: of human age groups Sialic Red York, Cell application Marikovsky the surface Acad A: Mobility 33:159-163, red (ed): Blood l975,pp 1961 Yaari 8. E: extruded New Marikovsky et #{227}gCs. CR 7. B and pp 23-38 charge scope Skutelsky cells Ramot jeunes different red Metabolism. Danon 6. Y, of old El Aspartate -45, 1969 amino- From www.bloodjournal.org by guest on August 9, 2017. For personal use only. RED 1011 CELL AGING transferase (GOT) erythrocytes. 34. the Seaman Supramol Med Struct D: The and Schauer Danon young Clin GVF: erythrocyte 35. from I Lab AP, of membranes. Chem 36. of 5, 37. acid Sper 38. G: 36:447 39. Baxter A, Hanahan Cohen virus 1961 of Boll neur- Soc Ital of the Biophys iG: human Acta Di: Ekholm Biochemical human Acta 419:229-242, Changes in surface erythrocytes aged 3:134-136, JE, of 709, 1970 1976 Biochim of Acta Flory LL, 44. slightly dif- 112:146-153, sity of D: red cell, Br Y, red Lotan R, on cells. Exp 19:701- Lis and H, Sharon labeling young Cell of blood J Haematol agglutinin blood EA: Studies human Agglutination soybean human MP, ages. Steane III. of Marikovsky Danon 45. Ti, different N, Red MG, characterization erythrocytes. group 15, 1973 Counter-current cells Biophys populations denand Res old 99:453- 1976 La Celle Arkin PL, B: of Cell M, change Weed Shape. Udkow and Potential in RI, New FH, fragmentation interaction: shape in Bessis Kirkpatrick Membrane membrane anisms in 1975 Luthra FW: blood haemagglutination. cells Ca2’ Soc Trans NS, density-separated Biophys cell neuramini- by blood 25:207-2 Selby Biochim Greenwalt separated Rela- proteins Biochim ages. 43. 456, The H, of red Quantitative of 1966 Z 1960 Beeley of Biochem 40. blood EA: Effect on agglutination Walter ferent 1973 carbohydrate vivo. -449, membrane. 300:341-378, red Steane Br J Haematol antibodies. de- Measurement 7:464-475, red cells. RL: treatment distribution from acid: Ti, IV. Quantitative Determination in human iuliano erythrocyte acids FH: and dase M, 1975 Br i Haematol Manfredi aminic quantitative neuraminic to haemolysis interaction. J H oppe-Seylers Gardner Greenwalt haemagglutination. of Wember acylneuraminic erythrocyte tionship Biol for 356:1727-1732, Yachnin human 41. 1971 42. 1973 eryth human membrane. Corfield A micromethod old chemistry 1:437-447. termination Physiol surface thrombocyte R, rocyte and 78:736-745, the mech- senescent Leblond York, PF Springer, red (eds): 1973, pp 69-78 of 46. Elastic Smith behavior cyte membranes. BD, La of Celle PT, senescent Biophys La human Celle PL: erythro- i 17:28a, 1977 (Abstr) From www.bloodjournal.org by guest on August 9, 2017. For personal use only. 1977 50: 1001-1011 Red cell agins. I. Surface charge density and sialic acid content of density-fractionated human erythrocytes GV Seaman, RJ Knox, FJ Nordt and DH Regan Updated information and services can be found at: http://www.bloodjournal.org/content/50/6/1001.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.