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Lipid composition of human red cells of different ages
BIOCHIMICAET BIOPHYSICA ACTA BRA 55645
LIPID
COMPOSITION
OF HUMAN
RED
CELLS
OF DIFFERENT
AGES
CHRISTINE C. WINTERBOURN* AND R. D. BATT of Chemistry and Biochemistry, LWassey University, Palmerston North (New Zealand) (Received September 5th, 1969)
Department
SUMMARY The method developed by PIOMELLI et al. (J. Lab. Clin. Med., 6g (1967) 659) for fractionating red cells according to age by ultracentrifugation over a discontinuous density gradient of isotonic albumin solutions, has been slightly modified and adapted for use on a larger scale. The degree of age separation of rat red cells has been examined and found to be comparable to that achieved for rabbit cells by the original method. Using this fractionation technique, which appears to produce red cell populations with considerably different mean ages, variations in lipid content of human red cells of different ages have been examined. A small decrease in total lipid content with increasing age was detected. However, this decrease was most marked between the youngest and all the other cell fractions, and suggests that the main lipid changes which occur in aging red cells take place during the transition from reticulocytes to erythrocytes. No variations with age in the relative amounts of the major red cell lipids could be detected.
INTRODUCTION The mammalian red cell, with its inability to divide, or to synthesise proteins when mature, provides an interesting system for studies in cellular aging. The cell initially contains a full complement of enzymes and cofactors, but is capable of existing in the blood stream for only a predetermined time. Although there are some changes in structure, composition, and metabolic activity as red cells age+12, the direct causes of these changes, and of the ultimate removal of the cells from circulation, have yet to be defined. A major problem which arises in the study of red cell aging is the difficulty of separating cells of different ages from a normal circulating population. Fractions enriched with younger and older cells have been obtained by a variety of methods, most of which are based on the property that cell density increases with age1-3. After normal centrifugation, there is some concentration of younger cells at * Present address: Division of Neurological Sciences, Department British Columbia,
Vancouver,
of Psychiatry,
University
of
Canada. Biochim. Biophys.
Acta, zoz (1970)
1-8
C. C. WINTERBOURN,
2
R. D. BATT
the top, and older cells at the bottom of the packed cell co1umn13s1’.The separation is slightly enhanced by ultracentrifugation 15, but what at present appears to give the best separation of red cells according to age is ultracentrifugation in a discontinuous gradient of isotonic albumin solutions l6. With this method there is equilibration of the cells in discrete bands between the albumin solutions. Studies on red cells situated at different positions in the cell column after centrifugation, either at low7 or high speeds8, suggest that red cell lipid content decreases with cell age. Since the lipids are almost certainly all membrane-localisedl7 lipid loss could lead to membrane malfunction and thus be involved in the aging process. In the present study, the albumin gradient ultracentrifugation technique has been used to more accurately assess changes in lipid concentration with red cell age. MATERIALS AND METHODS Red cell fractionation
technique
The method was basically that of PIOMELLI et aZ.le, modified for use on a larger scale and with human or rat cells. Some simplifications were also introduced. Density gradients were prepared from solutions of bovine serum albumin, Cohn Fraction V (Sigma Chemical Co.) in isotonic saline, rather than solutions of crystalline albumin in buffered saline containing glucose. No hemolysis of denser erythrocytes in the presence of Fraction V albumin, as experienced by PIOMELLI et aLIe, was observed, perhaps because of differences in the two commercial preparations. It was possible to reutilise the albumin after cell fractionation, by dialysing the solution free of salts, and then freeze-drying. No adverse effects on cell separations resulted from this procedure. Albumin was dissolved in water to give approx. a 40% solution. The osmolality*, density and albumin concentration of the solution were measured, and the osmolality made up to 290 mosM by adding solid NaCl. This solution was diluted with 0.92% NaCl solution (290 mosM) to give six solutions with densities ranging between 1.075 and 1.100, (albumin concentrations, 30-40%). The lowest density was intermediate between those of red and white blood cells, and the highest was just greater than that of the heaviest red cells. Gradients were prepared, at 4’, in either 6 or 30 ml cellulose * Osmolalities were measured with a Fiske osmometer. Measurements could not be made on the concentrated albumin solutions, because of freezing problems. When measurements on serial dilutions with water were made, the higher the dilution, the lower was the apparent osmolality of the original solution. This suggested that some interaction between albumin molecules was contributing to the measured osmolality value. This behaviour should be described by the relationship (M
M = T + ac2 = measured osmolality
and for a series of dilutions,
;
T = true osmolality
MD = T,+aDc2 (D = water dilution factor; MD and Dc2 was observed
; a = constant;
c = albumin
concentration),
T, = osmolality of undiluted albumin). A linear relationship between for all of the albumin solutions examined, regardless of salt concentration. In addition, when osmolalities calculated in this way were used to estimate the amount of NaCl required to bring a solution to a desired osmolality, good agreement between the predicted amount and that actually required was obtained. (When no correction was made, these values varied widely.) Osmolalities of concentrated albumin solutions were therefore calculated from measurements on serial dilutions using this relationship. Biochinz.
l?io#hys.
Acta,
202
(1970)
1-8
LIPIDS HUMAN
RED
CELLS
DIFFERENT
AGES
3
nitrate centrifuge tubes. Cell suspensions (hematocrit approx. 75%) were carefully layered over the albumin, and the tubes centrifuged at 4’, for 30 min at 25000 rev./min in a Spinco Model L ultracentrifuge, with either an SW3g or SW25 head. Up to 3 ml of packed cells could be applied to the larger tubes without overloading. With the density range of an entire red cell population being approx. 0.01 g/ml, small variations in density of the albumin layers can alter considerably the distribution of the cells in the gradient. Such variations can easily arise as a result of errors in density measurements (accuracy + 0.0005 g/ml), slight mixing during gradient preparation, or small temperature variations. Some density variations between different red cell populations are also possible. For these reasons, it is very difficult to design a system yielding five equal bands of red cells and accordingly, a small-scale preliminary run was always performed before a large-scale fractionation. If an unequal cell distribution was evident, solution densities were modified, and the probability of obtaining bands of cells of similar sizes was greatly increased. After centrifugation, the different red cell bands were collected separately by connecting the top of the centrifuge tube to a variable pressure head, piercing the bottom of the tube with a syringe needle, and allowing the contents to drip out. Clearly defined bands were cleanly separated, provided the drop rate was slow enough to prevent streaming down the centre of the tube, and the temperature was kept constant to prevent convective mixing. Cells were separated from albumin by mixing with an equal volume of isotonic saline, and centrifuging for IO min at 4000 rev. /min. After washing twice with isotonic saline, the cells were ready for further study. Examination of the albumin gradient technique for separating rat red cells according to age Rat blood (4 ml) was collected into heparin, and incubated with [2-Xlglycine (IO ,uC) (Radiochemical Centre, Amersham, specific activity 21.5 mC/mmole) at 37’ for 4 h. The red cells were separated, washed once with cold 0.9% saline, and resuspended in saline (hematocrit approx. 75%). Of the [z-Xlglycine, 36% was incorporated into the red cells; 38% of this was associated with the cellular protein. The red cell suspension, containing 1.6 ml packed cells and 0.5 ,LJCradioactivity in the cellular protein, was reinjected into a branch of the jugular vein of the same rat. Blood samples (1-2 ml) were withdrawn from the tail of the rat at intervals during the next 55 days. Washed red cells were fractionated by the albumin gradient procedure, using 3 ml tubes and spinning for 30 min at 25000 rev./min. Hemoglobin, total radioactivity, and protein radioactivity (precipitated with trichloroacetic acid) were estimated in the normal cell population and in each fraction. Radioactivity measurements were made with a Packard Model 2000 scintillation counter. Sample preparation was similar to the method of MAHIN AND LOFBERG'~, except that after digestion, 15 ml scintillation solution containing 2,5-diphenyloxazole (5 g) in a mixture of toluene (600 ml) and ethanol (400 ml) was added. The counting efficiency was approx. 45%, and there was no detectable glycine loss during digestion. of variations in lipid composition with cell age Blood was collected from healthy human donors (male and female, aged 20-35) into heparinised tubes. Red cells were separated, and washed twice in isotonic saline, Measurement
Biochim.
Biophys.
Acta,
202 (1970)
1-8
C. C. WI~TE~BO~R~, R. D. BATT
4
by centrifuging in a swingout head at 4”, at 3000 rev./min for 30 min. After each centrifugation, most of the upper plasma layer was removed. The remaining plasma was gently stirred to resuspend most of the white cells from the top of the cell column, and these were removed with a Pasteur pipette. The remaining cells were resuspended in saline and separated into five fractions by albumin gradient centrifugation. Hemoglobin and white cell concentrations were estimated for each fraction. In some studies, lipids were extracted from the intact cells from each fraction; in others, lipid concentrations were measured in conjunction with studies on the uptake of labelled plasma fatty acids into the cell ghostsf9. Ghosts were prepared by cell lysis in an equal volume of water. They were incubated in a plasma-Krebs-Ringer medium, and recovered and washed by centrifuging for 30 min at 13000rev./min. Supernatants were recentrifuged to check ghost recoveries. Lipids were extracted from red cells and ghosts, under N,, by the method of ROSE AND OKLANDER~, as described in a previous reportzl. Extracts were dissolved in chloroform-methanol (z:I, by vol.) for analysis, and stored under N, at -15~. Phospholipids were separated in duplicate by thin-layer chromatography on Merck silica gel G, with chloroform-methanol-acetic acid-water (65 : 25 : 8 : 4, by vol.) as solvent. The plates were activated at IIO' for I h, predeveloped in ethyl ether to remove impurities, reactivated at least 0.5 h, and used within 15 min of activation. Lipids were visualised with iodine, and phosphorus analyses were carried out without prior elution from the gel. Hemoglobin was determined by the cyanmethetnoglobin methodzz. Cell numbers were calculated from llemoglobin concentrations, by determining the amount of hemoglobin per unit volume of packed cells from a normal population, and taking a standard value23 for the packed cell volume of human red cells. White cells were counted after dilution in IO/~acetic acid. Total lipid was analysed by a modification of the method of AME~TTA~*,as described previously 2l. Results were read from a curve corresponding to a total lipid composition of 27% cholesterol and 733’; phospholipid. Lipid phosphorus was determined by the method of BARTLETT~~.When analyses were carried out on samples adsorbed to silica gel, 1.5 times the stated quantity of HCIO, was added, to prevent the development of an intense blue product with the reagents alone. Cholesterol was determined by the method of ABELL ct a1.26. RESULTS AND DISCUSSION
red cells byultraThe modified technique of PIOMELLI et al. 18, for fractionating centrifugation in a discontinuous gradient of isotonic albumin solutions, yielded five clearly distinguishable bands of red cells. The intervals between the bands contained only very low concentrations of cells. Similar results were obtained for both large and small scale separations. The densities of albumin solutions required for separation of rat cells were in the range r.ogo-x.075. White cells were concentrated above the lightest solution. The results of a study in which most of the white cells had been removed prior to fractionation indicated that 90% of the remainder were in this layer, with the others in the top red cell band (i.e., only O.I-0.5% of the original white cells were present in red cell fractions). The albumin solutions required for human red cell fractionation fell within the Biochim.
Biophys.
Acta,
mz
(1970)
I-S
LIPIDS HUMAN RED CELLS DIFFERENT AGES
5
density range x.ogo-1.078. The behaviour of human white cells was unexpected, in that instead of collecting above the d = 1.075 solution, they tended to concentrate in a band lying within the density range of the red cells. This phenomenon was independent of whether or not most of the white cells were removed prior to fractionation, and consequently could not be attributed to a small atypical fraction of cells. The most likely explanation appears to be that changes in white cell density were caused by either ingestion or adsorption of albumin molecules. The difficulties presented by the comparable positioning of red and white cells were minimised by prior removal of the bulk of the white cells. In addition, if the highest albumin density just exceeded red cell densities, the heavier white cells sedimented completely. Under these conditions, white cell contamination of red cell preparations was reduced to a very low level. Distribution of rat red cells of various ages, labelled with [G”C]glycine, im the albumin gradient Reticulocytes are the only circulating red cells capable of protein synthesiP7, so that on incubating blood with [z-Xlglycine, reticulocyte protein is the only red cell protein which becomes labelled. In addition, when the blood (from which the white cells have been removed), containing labelled reticulocytes, is readministered to the donor, insignificant amounts of label become incorporated into protein in other red cellP. Hence this procedure is very suitable for following the distribution of cells of a particular age in the albumin gradient. Red cells were withdrawn from a rat after administration of blood containing labelled reticulocytes, separated into five fractions, and the protein radioactivity in each fraction determined. The results, given in Fig. I, show that most of the red cells of each particular age were localised in a fairly narrow zone in the albumin gradient. Hence the method was effective in separating rat red cells into fractions with different mean ages. The degree of separation achieved is similar to that obtained by PIOMELLI et al.le for rabbit red cells. These authors have proposed a model which fits their results, and used this to calculate the mean age of each fraction. With rabbit cells (lifespan 60-70 days) they have calculated that the mean ages of five fractions should range from 9 to 51 days.
Day 0
Day 5
Day 55
ii
,
02466
I
I
,
Red cell
,
protein
0-
1
mdiooctivit:;
2
,
012
I
disint./min
a
( medn disint./min
Fig. 1. Discontinuous albumin gradient ultracentrifugation ministration of reticulocytes labelled with [z-l*C]glycine.
0-
1
2
per g hemoglobin per g hemoglobin >
of rat red cells at intervals after ad-
B&him.
Biophys.
Acta,
202
(1970)
1-8
6
C. C. WINTERBOURN,
R. D. BATT
The present study on red cell lipid composition has been carried out on human cells. An examination of the separation of human cells by this technique was not made, as it was felt that the administration of 14C-labelled cells to human subjects for this purpose was not justifiable. However, it is unlikely that the extent of age separation would differ substantially from that achieved for rabbit and rat. Centrifugation of red cells from any of these species produces an excess of young cells at the top and old cells at the bottom of the cell column, and the albumin gradient technique would be expected to improve this age separation for human cells, as it does for the others. On the assumption that a similar degree of separation is achieved for rabbit and human cells, from the data of PIOMELLI et aZ.IE it can be estimated that the mean ages of five bands of human cells (lifespan IZO days) should range from 15 to IOO days. With the achievement of such a separation, it should be possible to detect any variability of cell properties with age. Likewise any parameter which shows iittle or no variation between fractions would be unlikely to vary with cell age. Lipid composition of kwnnn red cells ofdi#erent ages Human red cells have been separated into fractions with different density, and the lipids from the cells or ghosts extracted and analysed. Mean values for the concentrations of total lipid, phospholipid, and cholesterol in each fraction are given in Table 1. No large variations between age fractions are apparent. The overall trend is a slight decrease with cell age in the amount of all lipid classes, but the biggest difference lies between the youngest and all the other fractions. The ratio of cholesterol to phospholipid was constant in all fractions. TABLE
1
LIPID COfjTENT OF FRACTIONSOF HUMAN RED CELLS WlTH DIFFERENT MEAPj AGES Red cells from each donor were separated into five fractions by ultracentrifugation in a discontinuous albumin gradient. Lipid concentrations in each fraction were measured. From graphs of the concentration of each lipid plotted against the cumulative percentage of cells from the top of the density gradient, concentrations in fractions corresponding to those in this table were determined. As most of the top fractions were less than 20% of the total cells, it was possible to determine lipid concentrations in the top Io”/b of the cells. The mean values for each fraction were calculated, and these (-_i S.B.) are quoted in the table. ~_______ --ChoEesicrol Frac- Cumulative &Y- Total ii-bid Phosbholibid ‘ 1" (ymoiesI~~ 1010 celk) (mg/I. 1010 cells) tima crat ofcells from ("g/T.'0 ..-_-l_l ---______ cells) No. the top of the Cells C&S Ghosts Ghosts dmsity gradient Cells ___..__... -_-..I O-IO 4.I5 rt 0.32 3.75 rt 0.24 4.02 i 0.30 1.05 * 0.03 0.99 * 0.12 2 IO-20 3.65 * 0.16 g.jO c”, 0.26 3.88 * 0.25 3 20-40 3.60 & 0.15 3.50 & 0.28 3.60 + 0.20 I.CO * 0.03 0.9r * 0.08 4 40-60 3.50 & O.II 3.45 & 0.18 3.56 * 0.14 1.00 f 0.03 0.90 & 0.06 5 60.-80 3.45 i 0.10 3.40 -& 0.07 3.51 & 0.20 o.Q5 * 0.03 0.91 * 0x9 6 80--r 00 3.40 i 0.05 3.30 i 0.05 3.60 zk 0.35 0.90 + 0.03 0.93 + 0.13 Number of individuals 3 4 3 2 3 _ _II__ 1
Data for ghosts and cells have been considered separately, because of the greater variability in ghost lipid measurements. In general, more total lipid was extracted from ghosts than from cells, and for this reason, no ghost total lipid measurements have been included. Additional ghost components were shown by thin-layer chromatography to be predominantly triglycerides and cholesterol esters. These lipid classes Biochim. Biophys. A&,
202 (1970) I-8
LIPIDS HUMAN RED CELLS DIFFERENT AGES
7
are only minor red cell constituents, and it is likely that the excess arose from the plasma in which the ghosts were incubated prior to extraction*. It is possible that ghosts could bind plasma lipids or lipoproteins strongly enough to withstand normal washing procedures. However, ghost and cell phospholipid and cholesterol concentrations agree quite closely, and both show similar relationships to cell age. No differences between the red cell fractions in the relative amounts of the major phospholipids could be detected (Table II). TABLE MAJOR
II PHOSPHOLIPIDS
Cumulative percentage of cells from top of centrifuged column O-20
Increasing Cell Age
20-40 40-60 _1 ;EIJ;,
IN
FRACTIONS
OF HUMAN
RED
CELLS
WITH
DIFFERENT
MEAN
AGES
Percent of Li$Gd phosphorus* Phosphatidylethanolamine* *
Phosphatidylcholine +phosphatidylserine***
Sphingonzyelin+
Others
29
46 44 44.5 43.5 43.5
24 25 24.5 25 25
I
ii.;
Means of four experiments. ** Includes both diacyl phosphoglycerides ** * These components were not completely dure employed. + Includes phosphatidylinositol.
I
I I I
*
and plasmalogens. separated by the thin-layer chromatographic
proce-
Using a fractionation method which should give good resolution of red cell age, it has been shown that there is very little difference in the lipid composition of young and old human red cells. No changes with age in the relative proportions of the major lipid components have been observed, and the only difference that is probably significant is the slightly higher total lipid concentration in the very youngest cells. In previous studies of changes in red cell lipids with age, WESTERMAN et aL7 and VAN GASTEL et aL8 have measured small differences in total lipid, phospholipid and cholesterol content between the top and bottom fractions of centrifuged human cells. Neither group detected any differences in phospholipid distribution. Variations measured in the present study are of a similar order to those obtained in both these investigations. In fact the enhanced age separation has revealed no greater differences between young and old cells, and has shown the biggest difference in lipid content to be between the very youngest and the remaining cells. As reticulocytes are considerand contain more lipid*a, it would appear that the ably larger than erythrocytes’, transition from reticulocyte to erythrocyte could account for the observed lipid difference between young and old red cells. The dissolution of the internal membranous structures of the reticulocyte would presumably be responsible for the decrease. The cell fractionation methods used by WESTERMAN et a1.7, VAN GASTEL et aL8, and PIOMELLI et a1.16 as used in the present study, although differing in ability to separate erythrocytes according to age, would all localise reticulocytes in the top fraction of cells. That differences between fractions in cell lipid concentration were similar in the three studies further suggests that changes in lipid content with red cell age only occur during maturation of reticulocytes. * These ghosts were used for the studies described in ref.
19.
Biochim. Biophys.
Acta, 202 (1970) 1-8
8
C. C. WINTERBOURN,
R. D. BATT
It seems unlikely that changes in lipid composition in aging red cells cause major modifications to cell structure and function. The difference in appearance of young and old cell membranes, observed by DANON AND PERKY, more likely arises from conformational changes involving reorganisation of interacting lipids and proteins, rather than differences in lipid composition. ACKNOWLEDGEMENTS
The authors would like to thank the Palmerston North Medical Research Foundation for a grant supporting this investigation, the staff of the Palmerston North Hospital Laboratory who collected the blood, and our colleagues who willingly donated their blood. REFERENCES I D. CHALFIN, J. Cellular Comp. Physiol., 47 (1956) 215. 2 J. F. HOFFMAN, J. CeUular Camp. Physiol., 51 (1958) 415. 3 T. A. J. PRANKERD, J. Physiol. London, 143 (1958) 325. 4 D. DANON AND K. PERK, J. Cellular Camp. Physiol., 59 (1962) 117. 5 Y. MARIKOVSKY, D. DANON AND A. KATCHALSKY, Biochim. Biophys. Acta, 124 (1966) 154. 6 H. WALTER, R. WINGE AND F. W, SELBY, Biochim. Biophys. Acta, log (1965) 293. 7 M. P. WESTERMAN, L. E. PIERCE AND W. N. JENSEN, J. Lab. C&z. Med., 62 (1963) 394. 8 C. VAN GASTEL, D. VAN DEN BERG, J. DE GIER AND L. L. M. VAN DEENEN, Brit. J. Haematol., 11 (1965) ‘93.
9 10 II 12
B. G. H. P.
L. W. D. A.
WALICER AND M. YURKOWSKI, Biochem. J., 103 (1967) L~HR APED H. D. WALLER, Folia Haematol., 78 (1962) WALLER, Stand. J. Haematol. Ser. Haematol., 2 (1965) MARKS, A. B. JOHNSON, E. HIRSCHBERG AND J. BANKS,
218. 385. 34.
Ann. N.Y. Acad. Sci., 75 (1958)
95.
13 T. C. PRENTICE AND C. BISHOP, J, Cellular Comp. Physiol., 65 (1965) 113. 14 J. C. DREYFUS, G. SHAPIRA AND J. KRUH, Compt. Rend. Sot. Biol., 144 (1950) 792. 15 D. A. RIGAS AND R. D. KOLER, J. Lab. Clin. Med., 58 (1961) 242. 16 S. PIOMELLI, G. LURINSKY AND L. R. WASSERMAN, J. Lab. Clin. Med., 69 (1967) 659. I7 J. T. DODGE, C. MITCHELL AXD D. J. HANAHAN, Arch. Biochem. Biophys., IOO (1963) 119. 18 D. T. MAHIN AND R. T. LOFBERG, Anal. Biochem., 16 (1966) 500. Ig C. C. WINTERBOURN AND R. D. BATT Biochim. Biophys. Acta 202 (1970) 9. 20 H. G. ROSE AND M. OKLANDER, J. Lipid Res., 6 (1965) 428. 21 C. C. WINTERBOURK AND R. D. BATT, Biochim. Biophys. Acta, 152 (1968) 255. 22 I. DAVIDSOHN AND B. B. WELLS, Clinical Diagnosis by Laboratory Methods, Saunders, Philadelphia, 1962, p. 73. 23 M. M. WINTROBE, Clinical Haematology, Kimpton, London, 5th ed., 1961, p. 95. 24 J. S. AMENTA, J. Lipid Res., 5 (1964) 270. 25 G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. 26 L. L. ABELL, B. B. LEVY, B. B. BRODIE AND F. E. KENDALL, J. Viol. Chem., 195 (1952) 357. 27 D. W. ALLEN, in C. BISHOP AND D. M. SURGENOR, The Red Blood Cell, Academic Press, New York, 1964, p. 309. 28 H. J. RADERECHT, E. SCHGLZEI. AND S. M. RAPOPORT, Klin. E’ochschr., 38 (1960) 824. BiOChim. Biophys.
Acta, 202 (1970) I-8
LIPID
COMPOSITION
OF HUMAN
RED
CELLS
OF DIFFERENT
AGES
CHRISTINE C. WINTERBOURN* AND R. D. BATT of Chemistry and Biochemistry, LWassey University, Palmerston North (New Zealand) (Received September 5th, 1969)
Department
SUMMARY The method developed by PIOMELLI et al. (J. Lab. Clin. Med., 6g (1967) 659) for fractionating red cells according to age by ultracentrifugation over a discontinuous density gradient of isotonic albumin solutions, has been slightly modified and adapted for use on a larger scale. The degree of age separation of rat red cells has been examined and found to be comparable to that achieved for rabbit cells by the original method. Using this fractionation technique, which appears to produce red cell populations with considerably different mean ages, variations in lipid content of human red cells of different ages have been examined. A small decrease in total lipid content with increasing age was detected. However, this decrease was most marked between the youngest and all the other cell fractions, and suggests that the main lipid changes which occur in aging red cells take place during the transition from reticulocytes to erythrocytes. No variations with age in the relative amounts of the major red cell lipids could be detected.
INTRODUCTION The mammalian red cell, with its inability to divide, or to synthesise proteins when mature, provides an interesting system for studies in cellular aging. The cell initially contains a full complement of enzymes and cofactors, but is capable of existing in the blood stream for only a predetermined time. Although there are some changes in structure, composition, and metabolic activity as red cells age+12, the direct causes of these changes, and of the ultimate removal of the cells from circulation, have yet to be defined. A major problem which arises in the study of red cell aging is the difficulty of separating cells of different ages from a normal circulating population. Fractions enriched with younger and older cells have been obtained by a variety of methods, most of which are based on the property that cell density increases with age1-3. After normal centrifugation, there is some concentration of younger cells at * Present address: Division of Neurological Sciences, Department British Columbia,
Vancouver,
of Psychiatry,
University
of
Canada. Biochim. Biophys.
Acta, zoz (1970)
1-8
C. C. WINTERBOURN,
2
R. D. BATT
the top, and older cells at the bottom of the packed cell co1umn13s1’.The separation is slightly enhanced by ultracentrifugation 15, but what at present appears to give the best separation of red cells according to age is ultracentrifugation in a discontinuous gradient of isotonic albumin solutions l6. With this method there is equilibration of the cells in discrete bands between the albumin solutions. Studies on red cells situated at different positions in the cell column after centrifugation, either at low7 or high speeds8, suggest that red cell lipid content decreases with cell age. Since the lipids are almost certainly all membrane-localisedl7 lipid loss could lead to membrane malfunction and thus be involved in the aging process. In the present study, the albumin gradient ultracentrifugation technique has been used to more accurately assess changes in lipid concentration with red cell age. MATERIALS AND METHODS Red cell fractionation
technique
The method was basically that of PIOMELLI et aZ.le, modified for use on a larger scale and with human or rat cells. Some simplifications were also introduced. Density gradients were prepared from solutions of bovine serum albumin, Cohn Fraction V (Sigma Chemical Co.) in isotonic saline, rather than solutions of crystalline albumin in buffered saline containing glucose. No hemolysis of denser erythrocytes in the presence of Fraction V albumin, as experienced by PIOMELLI et aLIe, was observed, perhaps because of differences in the two commercial preparations. It was possible to reutilise the albumin after cell fractionation, by dialysing the solution free of salts, and then freeze-drying. No adverse effects on cell separations resulted from this procedure. Albumin was dissolved in water to give approx. a 40% solution. The osmolality*, density and albumin concentration of the solution were measured, and the osmolality made up to 290 mosM by adding solid NaCl. This solution was diluted with 0.92% NaCl solution (290 mosM) to give six solutions with densities ranging between 1.075 and 1.100, (albumin concentrations, 30-40%). The lowest density was intermediate between those of red and white blood cells, and the highest was just greater than that of the heaviest red cells. Gradients were prepared, at 4’, in either 6 or 30 ml cellulose * Osmolalities were measured with a Fiske osmometer. Measurements could not be made on the concentrated albumin solutions, because of freezing problems. When measurements on serial dilutions with water were made, the higher the dilution, the lower was the apparent osmolality of the original solution. This suggested that some interaction between albumin molecules was contributing to the measured osmolality value. This behaviour should be described by the relationship (M
M = T + ac2 = measured osmolality
and for a series of dilutions,
;
T = true osmolality
MD = T,+aDc2 (D = water dilution factor; MD and Dc2 was observed
; a = constant;
c = albumin
concentration),
T, = osmolality of undiluted albumin). A linear relationship between for all of the albumin solutions examined, regardless of salt concentration. In addition, when osmolalities calculated in this way were used to estimate the amount of NaCl required to bring a solution to a desired osmolality, good agreement between the predicted amount and that actually required was obtained. (When no correction was made, these values varied widely.) Osmolalities of concentrated albumin solutions were therefore calculated from measurements on serial dilutions using this relationship. Biochinz.
l?io#hys.
Acta,
202
(1970)
1-8
LIPIDS HUMAN
RED
CELLS
DIFFERENT
AGES
3
nitrate centrifuge tubes. Cell suspensions (hematocrit approx. 75%) were carefully layered over the albumin, and the tubes centrifuged at 4’, for 30 min at 25000 rev./min in a Spinco Model L ultracentrifuge, with either an SW3g or SW25 head. Up to 3 ml of packed cells could be applied to the larger tubes without overloading. With the density range of an entire red cell population being approx. 0.01 g/ml, small variations in density of the albumin layers can alter considerably the distribution of the cells in the gradient. Such variations can easily arise as a result of errors in density measurements (accuracy + 0.0005 g/ml), slight mixing during gradient preparation, or small temperature variations. Some density variations between different red cell populations are also possible. For these reasons, it is very difficult to design a system yielding five equal bands of red cells and accordingly, a small-scale preliminary run was always performed before a large-scale fractionation. If an unequal cell distribution was evident, solution densities were modified, and the probability of obtaining bands of cells of similar sizes was greatly increased. After centrifugation, the different red cell bands were collected separately by connecting the top of the centrifuge tube to a variable pressure head, piercing the bottom of the tube with a syringe needle, and allowing the contents to drip out. Clearly defined bands were cleanly separated, provided the drop rate was slow enough to prevent streaming down the centre of the tube, and the temperature was kept constant to prevent convective mixing. Cells were separated from albumin by mixing with an equal volume of isotonic saline, and centrifuging for IO min at 4000 rev. /min. After washing twice with isotonic saline, the cells were ready for further study. Examination of the albumin gradient technique for separating rat red cells according to age Rat blood (4 ml) was collected into heparin, and incubated with [2-Xlglycine (IO ,uC) (Radiochemical Centre, Amersham, specific activity 21.5 mC/mmole) at 37’ for 4 h. The red cells were separated, washed once with cold 0.9% saline, and resuspended in saline (hematocrit approx. 75%). Of the [z-Xlglycine, 36% was incorporated into the red cells; 38% of this was associated with the cellular protein. The red cell suspension, containing 1.6 ml packed cells and 0.5 ,LJCradioactivity in the cellular protein, was reinjected into a branch of the jugular vein of the same rat. Blood samples (1-2 ml) were withdrawn from the tail of the rat at intervals during the next 55 days. Washed red cells were fractionated by the albumin gradient procedure, using 3 ml tubes and spinning for 30 min at 25000 rev./min. Hemoglobin, total radioactivity, and protein radioactivity (precipitated with trichloroacetic acid) were estimated in the normal cell population and in each fraction. Radioactivity measurements were made with a Packard Model 2000 scintillation counter. Sample preparation was similar to the method of MAHIN AND LOFBERG'~, except that after digestion, 15 ml scintillation solution containing 2,5-diphenyloxazole (5 g) in a mixture of toluene (600 ml) and ethanol (400 ml) was added. The counting efficiency was approx. 45%, and there was no detectable glycine loss during digestion. of variations in lipid composition with cell age Blood was collected from healthy human donors (male and female, aged 20-35) into heparinised tubes. Red cells were separated, and washed twice in isotonic saline, Measurement
Biochim.
Biophys.
Acta,
202 (1970)
1-8
C. C. WI~TE~BO~R~, R. D. BATT
4
by centrifuging in a swingout head at 4”, at 3000 rev./min for 30 min. After each centrifugation, most of the upper plasma layer was removed. The remaining plasma was gently stirred to resuspend most of the white cells from the top of the cell column, and these were removed with a Pasteur pipette. The remaining cells were resuspended in saline and separated into five fractions by albumin gradient centrifugation. Hemoglobin and white cell concentrations were estimated for each fraction. In some studies, lipids were extracted from the intact cells from each fraction; in others, lipid concentrations were measured in conjunction with studies on the uptake of labelled plasma fatty acids into the cell ghostsf9. Ghosts were prepared by cell lysis in an equal volume of water. They were incubated in a plasma-Krebs-Ringer medium, and recovered and washed by centrifuging for 30 min at 13000rev./min. Supernatants were recentrifuged to check ghost recoveries. Lipids were extracted from red cells and ghosts, under N,, by the method of ROSE AND OKLANDER~, as described in a previous reportzl. Extracts were dissolved in chloroform-methanol (z:I, by vol.) for analysis, and stored under N, at -15~. Phospholipids were separated in duplicate by thin-layer chromatography on Merck silica gel G, with chloroform-methanol-acetic acid-water (65 : 25 : 8 : 4, by vol.) as solvent. The plates were activated at IIO' for I h, predeveloped in ethyl ether to remove impurities, reactivated at least 0.5 h, and used within 15 min of activation. Lipids were visualised with iodine, and phosphorus analyses were carried out without prior elution from the gel. Hemoglobin was determined by the cyanmethetnoglobin methodzz. Cell numbers were calculated from llemoglobin concentrations, by determining the amount of hemoglobin per unit volume of packed cells from a normal population, and taking a standard value23 for the packed cell volume of human red cells. White cells were counted after dilution in IO/~acetic acid. Total lipid was analysed by a modification of the method of AME~TTA~*,as described previously 2l. Results were read from a curve corresponding to a total lipid composition of 27% cholesterol and 733’; phospholipid. Lipid phosphorus was determined by the method of BARTLETT~~.When analyses were carried out on samples adsorbed to silica gel, 1.5 times the stated quantity of HCIO, was added, to prevent the development of an intense blue product with the reagents alone. Cholesterol was determined by the method of ABELL ct a1.26. RESULTS AND DISCUSSION
red cells byultraThe modified technique of PIOMELLI et al. 18, for fractionating centrifugation in a discontinuous gradient of isotonic albumin solutions, yielded five clearly distinguishable bands of red cells. The intervals between the bands contained only very low concentrations of cells. Similar results were obtained for both large and small scale separations. The densities of albumin solutions required for separation of rat cells were in the range r.ogo-x.075. White cells were concentrated above the lightest solution. The results of a study in which most of the white cells had been removed prior to fractionation indicated that 90% of the remainder were in this layer, with the others in the top red cell band (i.e., only O.I-0.5% of the original white cells were present in red cell fractions). The albumin solutions required for human red cell fractionation fell within the Biochim.
Biophys.
Acta,
mz
(1970)
I-S
LIPIDS HUMAN RED CELLS DIFFERENT AGES
5
density range x.ogo-1.078. The behaviour of human white cells was unexpected, in that instead of collecting above the d = 1.075 solution, they tended to concentrate in a band lying within the density range of the red cells. This phenomenon was independent of whether or not most of the white cells were removed prior to fractionation, and consequently could not be attributed to a small atypical fraction of cells. The most likely explanation appears to be that changes in white cell density were caused by either ingestion or adsorption of albumin molecules. The difficulties presented by the comparable positioning of red and white cells were minimised by prior removal of the bulk of the white cells. In addition, if the highest albumin density just exceeded red cell densities, the heavier white cells sedimented completely. Under these conditions, white cell contamination of red cell preparations was reduced to a very low level. Distribution of rat red cells of various ages, labelled with [G”C]glycine, im the albumin gradient Reticulocytes are the only circulating red cells capable of protein synthesiP7, so that on incubating blood with [z-Xlglycine, reticulocyte protein is the only red cell protein which becomes labelled. In addition, when the blood (from which the white cells have been removed), containing labelled reticulocytes, is readministered to the donor, insignificant amounts of label become incorporated into protein in other red cellP. Hence this procedure is very suitable for following the distribution of cells of a particular age in the albumin gradient. Red cells were withdrawn from a rat after administration of blood containing labelled reticulocytes, separated into five fractions, and the protein radioactivity in each fraction determined. The results, given in Fig. I, show that most of the red cells of each particular age were localised in a fairly narrow zone in the albumin gradient. Hence the method was effective in separating rat red cells into fractions with different mean ages. The degree of separation achieved is similar to that obtained by PIOMELLI et al.le for rabbit red cells. These authors have proposed a model which fits their results, and used this to calculate the mean age of each fraction. With rabbit cells (lifespan 60-70 days) they have calculated that the mean ages of five fractions should range from 9 to 51 days.
Day 0
Day 5
Day 55
ii
,
02466
I
I
,
Red cell
,
protein
0-
1
mdiooctivit:;
2
,
012
I
disint./min
a
( medn disint./min
Fig. 1. Discontinuous albumin gradient ultracentrifugation ministration of reticulocytes labelled with [z-l*C]glycine.
0-
1
2
per g hemoglobin per g hemoglobin >
of rat red cells at intervals after ad-
B&him.
Biophys.
Acta,
202
(1970)
1-8
6
C. C. WINTERBOURN,
R. D. BATT
The present study on red cell lipid composition has been carried out on human cells. An examination of the separation of human cells by this technique was not made, as it was felt that the administration of 14C-labelled cells to human subjects for this purpose was not justifiable. However, it is unlikely that the extent of age separation would differ substantially from that achieved for rabbit and rat. Centrifugation of red cells from any of these species produces an excess of young cells at the top and old cells at the bottom of the cell column, and the albumin gradient technique would be expected to improve this age separation for human cells, as it does for the others. On the assumption that a similar degree of separation is achieved for rabbit and human cells, from the data of PIOMELLI et aZ.IE it can be estimated that the mean ages of five bands of human cells (lifespan IZO days) should range from 15 to IOO days. With the achievement of such a separation, it should be possible to detect any variability of cell properties with age. Likewise any parameter which shows iittle or no variation between fractions would be unlikely to vary with cell age. Lipid composition of kwnnn red cells ofdi#erent ages Human red cells have been separated into fractions with different density, and the lipids from the cells or ghosts extracted and analysed. Mean values for the concentrations of total lipid, phospholipid, and cholesterol in each fraction are given in Table 1. No large variations between age fractions are apparent. The overall trend is a slight decrease with cell age in the amount of all lipid classes, but the biggest difference lies between the youngest and all the other fractions. The ratio of cholesterol to phospholipid was constant in all fractions. TABLE
1
LIPID COfjTENT OF FRACTIONSOF HUMAN RED CELLS WlTH DIFFERENT MEAPj AGES Red cells from each donor were separated into five fractions by ultracentrifugation in a discontinuous albumin gradient. Lipid concentrations in each fraction were measured. From graphs of the concentration of each lipid plotted against the cumulative percentage of cells from the top of the density gradient, concentrations in fractions corresponding to those in this table were determined. As most of the top fractions were less than 20% of the total cells, it was possible to determine lipid concentrations in the top Io”/b of the cells. The mean values for each fraction were calculated, and these (-_i S.B.) are quoted in the table. ~_______ --ChoEesicrol Frac- Cumulative &Y- Total ii-bid Phosbholibid ‘ 1" (ymoiesI~~ 1010 celk) (mg/I. 1010 cells) tima crat ofcells from ("g/T.'0 ..-_-l_l ---______ cells) No. the top of the Cells C&S Ghosts Ghosts dmsity gradient Cells ___..__... -_-..I O-IO 4.I5 rt 0.32 3.75 rt 0.24 4.02 i 0.30 1.05 * 0.03 0.99 * 0.12 2 IO-20 3.65 * 0.16 g.jO c”, 0.26 3.88 * 0.25 3 20-40 3.60 & 0.15 3.50 & 0.28 3.60 + 0.20 I.CO * 0.03 0.9r * 0.08 4 40-60 3.50 & O.II 3.45 & 0.18 3.56 * 0.14 1.00 f 0.03 0.90 & 0.06 5 60.-80 3.45 i 0.10 3.40 -& 0.07 3.51 & 0.20 o.Q5 * 0.03 0.91 * 0x9 6 80--r 00 3.40 i 0.05 3.30 i 0.05 3.60 zk 0.35 0.90 + 0.03 0.93 + 0.13 Number of individuals 3 4 3 2 3 _ _II__ 1
Data for ghosts and cells have been considered separately, because of the greater variability in ghost lipid measurements. In general, more total lipid was extracted from ghosts than from cells, and for this reason, no ghost total lipid measurements have been included. Additional ghost components were shown by thin-layer chromatography to be predominantly triglycerides and cholesterol esters. These lipid classes Biochim. Biophys. A&,
202 (1970) I-8
LIPIDS HUMAN RED CELLS DIFFERENT AGES
7
are only minor red cell constituents, and it is likely that the excess arose from the plasma in which the ghosts were incubated prior to extraction*. It is possible that ghosts could bind plasma lipids or lipoproteins strongly enough to withstand normal washing procedures. However, ghost and cell phospholipid and cholesterol concentrations agree quite closely, and both show similar relationships to cell age. No differences between the red cell fractions in the relative amounts of the major phospholipids could be detected (Table II). TABLE MAJOR
II PHOSPHOLIPIDS
Cumulative percentage of cells from top of centrifuged column O-20
Increasing Cell Age
20-40 40-60 _1 ;EIJ;,
IN
FRACTIONS
OF HUMAN
RED
CELLS
WITH
DIFFERENT
MEAN
AGES
Percent of Li$Gd phosphorus* Phosphatidylethanolamine* *
Phosphatidylcholine +phosphatidylserine***
Sphingonzyelin+
Others
29
46 44 44.5 43.5 43.5
24 25 24.5 25 25
I
ii.;
Means of four experiments. ** Includes both diacyl phosphoglycerides ** * These components were not completely dure employed. + Includes phosphatidylinositol.
I
I I I
*
and plasmalogens. separated by the thin-layer chromatographic
proce-
Using a fractionation method which should give good resolution of red cell age, it has been shown that there is very little difference in the lipid composition of young and old human red cells. No changes with age in the relative proportions of the major lipid components have been observed, and the only difference that is probably significant is the slightly higher total lipid concentration in the very youngest cells. In previous studies of changes in red cell lipids with age, WESTERMAN et aL7 and VAN GASTEL et aL8 have measured small differences in total lipid, phospholipid and cholesterol content between the top and bottom fractions of centrifuged human cells. Neither group detected any differences in phospholipid distribution. Variations measured in the present study are of a similar order to those obtained in both these investigations. In fact the enhanced age separation has revealed no greater differences between young and old cells, and has shown the biggest difference in lipid content to be between the very youngest and the remaining cells. As reticulocytes are considerand contain more lipid*a, it would appear that the ably larger than erythrocytes’, transition from reticulocyte to erythrocyte could account for the observed lipid difference between young and old red cells. The dissolution of the internal membranous structures of the reticulocyte would presumably be responsible for the decrease. The cell fractionation methods used by WESTERMAN et a1.7, VAN GASTEL et aL8, and PIOMELLI et a1.16 as used in the present study, although differing in ability to separate erythrocytes according to age, would all localise reticulocytes in the top fraction of cells. That differences between fractions in cell lipid concentration were similar in the three studies further suggests that changes in lipid content with red cell age only occur during maturation of reticulocytes. * These ghosts were used for the studies described in ref.
19.
Biochim. Biophys.
Acta, 202 (1970) 1-8
8
C. C. WINTERBOURN,
R. D. BATT
It seems unlikely that changes in lipid composition in aging red cells cause major modifications to cell structure and function. The difference in appearance of young and old cell membranes, observed by DANON AND PERKY, more likely arises from conformational changes involving reorganisation of interacting lipids and proteins, rather than differences in lipid composition. ACKNOWLEDGEMENTS
The authors would like to thank the Palmerston North Medical Research Foundation for a grant supporting this investigation, the staff of the Palmerston North Hospital Laboratory who collected the blood, and our colleagues who willingly donated their blood. REFERENCES I D. CHALFIN, J. Cellular Comp. Physiol., 47 (1956) 215. 2 J. F. HOFFMAN, J. CeUular Camp. Physiol., 51 (1958) 415. 3 T. A. J. PRANKERD, J. Physiol. London, 143 (1958) 325. 4 D. DANON AND K. PERK, J. Cellular Camp. Physiol., 59 (1962) 117. 5 Y. MARIKOVSKY, D. DANON AND A. KATCHALSKY, Biochim. Biophys. Acta, 124 (1966) 154. 6 H. WALTER, R. WINGE AND F. W, SELBY, Biochim. Biophys. Acta, log (1965) 293. 7 M. P. WESTERMAN, L. E. PIERCE AND W. N. JENSEN, J. Lab. C&z. Med., 62 (1963) 394. 8 C. VAN GASTEL, D. VAN DEN BERG, J. DE GIER AND L. L. M. VAN DEENEN, Brit. J. Haematol., 11 (1965) ‘93.
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B. G. H. P.
L. W. D. A.
WALICER AND M. YURKOWSKI, Biochem. J., 103 (1967) L~HR APED H. D. WALLER, Folia Haematol., 78 (1962) WALLER, Stand. J. Haematol. Ser. Haematol., 2 (1965) MARKS, A. B. JOHNSON, E. HIRSCHBERG AND J. BANKS,
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13 T. C. PRENTICE AND C. BISHOP, J, Cellular Comp. Physiol., 65 (1965) 113. 14 J. C. DREYFUS, G. SHAPIRA AND J. KRUH, Compt. Rend. Sot. Biol., 144 (1950) 792. 15 D. A. RIGAS AND R. D. KOLER, J. Lab. Clin. Med., 58 (1961) 242. 16 S. PIOMELLI, G. LURINSKY AND L. R. WASSERMAN, J. Lab. Clin. Med., 69 (1967) 659. I7 J. T. DODGE, C. MITCHELL AXD D. J. HANAHAN, Arch. Biochem. Biophys., IOO (1963) 119. 18 D. T. MAHIN AND R. T. LOFBERG, Anal. Biochem., 16 (1966) 500. Ig C. C. WINTERBOURN AND R. D. BATT Biochim. Biophys. Acta 202 (1970) 9. 20 H. G. ROSE AND M. OKLANDER, J. Lipid Res., 6 (1965) 428. 21 C. C. WINTERBOURK AND R. D. BATT, Biochim. Biophys. Acta, 152 (1968) 255. 22 I. DAVIDSOHN AND B. B. WELLS, Clinical Diagnosis by Laboratory Methods, Saunders, Philadelphia, 1962, p. 73. 23 M. M. WINTROBE, Clinical Haematology, Kimpton, London, 5th ed., 1961, p. 95. 24 J. S. AMENTA, J. Lipid Res., 5 (1964) 270. 25 G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. 26 L. L. ABELL, B. B. LEVY, B. B. BRODIE AND F. E. KENDALL, J. Viol. Chem., 195 (1952) 357. 27 D. W. ALLEN, in C. BISHOP AND D. M. SURGENOR, The Red Blood Cell, Academic Press, New York, 1964, p. 309. 28 H. J. RADERECHT, E. SCHGLZEI. AND S. M. RAPOPORT, Klin. E’ochschr., 38 (1960) 824. BiOChim. Biophys.
Acta, 202 (1970) I-8