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Aging of human erythrocytes
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Aging Differential
Sensitivity
174, 463-468
of Human
(1976)
Erythrocytes
of Young and Old Erythrocytes to Hemolysis Peroxide in the Presence of Thyroxine
RICHARD
WALLS,
Department
ofPharmacology,
K. SREE
KUMAR,
University Los Angeles, Received
AND
ofsouthern California
November
PAUL
California 90033
Induced
by
HOCHSTEIN School
ofMedicine,
10, 1975
Human erythrocytes were separated according to cell age using albumin density gradients. In the presence of glucose (100 mg%), young cells were able to effectively protect themselves against thyroxine-peroxide induced hemolysis; old cells exhibited less protection. Hemolysis in heterogeneous populations is preceded by lipid peroxidation, K+ leak and decreased filtrability of the cells. Hydroxy radical scavengers partially inhibited hemolysis while superoxide dismutase had no effect. It is postulated that the differential sensitivity of young and old erythrocytes to thyroxine-peroxide induced metabolic and morphological alterations may play a role in the recognition and removal of senescent cells from the circulation.
Human erythrocytes have a finite life span of about 120 days. Their aging in vivo is accompanied by a decrease in the activity of enzymes such as glucose-6-phosphate dehydrogenase (G-6-PD)’ as well as by alterations in other cellular components (1). G-6-PD also plays a cardinal role in the detoxification of hydrogen peroxide (2). It is well established that red cells with a genetic deficiency of this enzyme undergo oxidative alterations when exposed to peroxide (3). We recently reported that the hemolysis of such cells in the presence of hydrogen peroxide was markedly enhanced by thyroid hormone (4). We suggested that thyroxine is a potentiating factor in the anemia associated with oxidative stress in G-6-PD-deficient individuals and that the toxicity of autoxidizable drugs is a consequence of both the formation of hydrogen peroxide and the availability of thyroid hormones. This concept raises the question of ’ Abbreviations used: G-6.PD, glucose-6-phosphate dehydrogenase: L-T,, L-thyroxine; BSG, buffered-saline-glucose; MA, malonaldehyde; TBA, Z-thiobarbituric acid; SOD, superoxide dismutase.
METHODS Hemolysis volunteers 463
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
whether thyroxine might also participate in the recognition and destruction of normal erythrocytes during the aging process. In this connection, McClellan et aZ. (5) have reported that the survival time of the erythrocytes of hyperthyroidic individuals is shortened, whereas the transfusion of these cells to euthyroid persons results in their normal survival. The experiments described in this paper were undertaken to determine whether or not older normal cells, which have decreased G-6-PD activity, exhibit an increased sensitivity to the cytotoxic effects initiated by peroxides in the presence of thyroxine. Our results demonstrate that, in fact, the response of older cells to these agents is similar to that of G-6-PD delicient cells and support the concept that thyroxine may be involved in the aging process. We also describe the nature of some of the prelytic events which may be relevant to the entrapment of senescent cells in the circulation. experiments. was collected
Blood from healthy, adult either into heparinized,
464
P
WALLS,
KUMAR
disposable syringes or defibrinized with glass beads. The cells were washed three times with 0.9% NaCl0.1% human albumin, and the final hematocrit was adjusted to 30%. The cells were utilized within 24 h. Erythrocytes (1% hematocrit) were exposed to a peroxide generating system (1.6 x lo-’ U/ml xanthine oxidase and 1.67 x 10m3 M hypoxanthine) with and without the inclusion of L-thyroxine (L-T,) in a final 3.0-ml reaction volume. A modified KrebsRinger phosphate buffer, pH 7.0, (48 mM K-PO,, pH 7.0; 73 mu NaCl; 5 mM KCl; 1.2 InM MgSO,.7 H,O; 0.9 mM CaC1.2 H,O) was used as the incubation medium. When glucose was included in the reaction mixture, the final concentration was 100 mg% (5.5 mM). L-Thyroxine was prepared in 0.05 N NaOH. All incubations were at 37°C with continuous shaking in lo- or 5-ml conical flasks. One-milliliter samples were withdrawn from incubation flasks at desired time intervals and centrifuged at 1OOOg for 5 min. Supernatant (0.65 ml) was then removed and mixed with 1.3 ml of Drabkin’s reagent (100 mg of NaCN and 300 mg of K,,Fe(CN),, in 1000 ml of H,O) (6), and the extent of hemolysis was determined at 540 nm using a Gilford Spectrophotometer. One hundred percent hemolysis was determined by adding 0.1 ml of 30% erythrocytes to 2.9 ml of glass-distilled H,O. This sample was then assayed for hemolysis as described above. Separation of old and young cells. The technique of Piomelli et al. (7) for rabbit cell fractionation was modified to suit human erythrocytes. Buffered-saline-glucose (BSG; 8.12 g NaCl, 1.22 g Na,HPO,, 0.219 g NaH,P0,,2H,O, 2.0 g glucose, made up to 1000 ml glass-distilled H,O) was used to wash the red cells obtained from defibrinated blood. The cells were then packed by centrifugation at 1OOOg for 5 min. A stock solution of 46 gYo albumin was prepared in water. The stock albumin solution was diluted with BSG to form three solutions with specific gravities of 1.087, 1.091, and 1.095. The gradient was prepared by layering equal volumes (ca. 3.0 ml) of each of the albumin solutions carefully into 12-m], round bottom, polypropylene tubes, starting with the most dense working up to the least. Packed cells (1.5-2.0 ml) were added to the top of the gradient. The tubes were then centrifuged at 2-4°C for 60 min at 39,000 rpm using an SB283 rotor in an IEC, Model B60, ultracentrifuge. The cells separated into four bands, including the bottom sediment and were aspirated into tubes from the top of the gradient by applying a small negative pressure. The cells were washed in saline and adjusted to a final 30% hematocrit with saline-albumin. The lowest band (fraction 4) corresponded to the older, most dense cells, whereas the top band (fraction 1) represented the younger cells. Glucose-&phosphate dehydrogenase (G-6-PD) activity was determined according to the method of Beutler (6).
AND
HOCHSTEIN
TBA reactive material. Malonaldehyde (MA) was determined in l.O-ml suspensions of 1.0% red cells. The l.O-ml sample was added to 0.1 ml of 50% trichloracetic acid and centrifuged at 1OOOg for 5 min to form a hard-packed pellet. The supernatant was removed and added to 0.5 ml 0.75% 2-thiobarbituric acid (TBA). Samples were boiled for 20 min and cooled to room temperature. Scans were made between 600 and 450 nm to determine the spectra of the chromogens. The MA-TBA chromogen has a maximum at 532 nm. Because of varying differences in background absorption, the MA formed was calculated by the difference in the readings obtained between 532 and 580 nm. K+ leak studies. The reaction mixture for hemolysis studies was used except that the buffer was replaced by 0.1 M Na-PO,, pH 7.0. After indicated time periods, the samples were centrifuged and 1.0 ml of the supernatant was removed. Glass-distilled deionized water (1.0 ml) was then added to the supernatant sample and the K+ was determined using a Perkin-Elmer Atomic Absorption Spectrophotometer, Model 290. All K+ determinations were made prior to the onset of hemolysis in order to assure the K’ loss as a prelytic event. Filtration studies. These studies were conducted using a Millipore filtration apparatus with 5-pm pore, 25-mm diameter Nucleopore membranes. The membranes were soaked in double-distilled water and sonicated prior to use. Cells were incubated in the presence of the peroxide generating system with or without 1 x lo-” M thyroxine for 1 h at 37°C. Filtration times were determined on 3.0-ml samples by recording the flow time of 1.0 ml after an initial 0.5 ml passed through the membrane. Xanthine oxidase (Sigma), hypoxanthine (Sigma), L-thyroxine (Nutritional Biochemicals), triiodothyronine (Calbiochem), and bovine albumin, Fraction V (Pentex, Miles Laboratories) were purchased. Superoxide dismutase (SOD) was prepared according to the method of McCord and Fridovich (8). One unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of cytochrome c by 50% at an initial rate of 0.5 ODI min. RESULTS
Differential sensitivity of old and young cells to glucose protection against hemolysis. The G-6-PD activity was determined
on the various cell fractions prepared on albumin gradients and compared to the activity of an unfractionated blood sample to assure age differences. The cells were then exposed to the peroxide generating system with or without L-thyroxine (lo-;’ M) and with or without 100 mg% glucose.
THYROXINE
AND
AGING
The amount of hemolysis was determined after 21/z h incubation at 37°C. The results are shown in Table I. The data, with the exception of the G-6-PD activity, are expressed as the percentage of hemolysis obtained. It is clear that in the absence of glucose, all cell populations are susceptible to hemolysis when exposed to the peroxide generating system and L-T,. In the presence of glucose, the younger cells are more able to protect themselves against hemolysis; whereas the older cells exhibited less protection. In the presence of glucose, unfractionated and young cells exhibited 75 to 85% protection against hemolysis, however the older cells exhibited only 25% protection. Thyroxine concentrations. The effect of various thyroxine concentrations and the peroxide generating system on a normal, heterogeneous population of cells is illustrated in Fig. 1. The protective effect of glucose (100 mg%) is also demonstrated. Hemolysis was assayed after 3 and 5 h incubation at 37°C. The results indicate that under the conditions of these experiments, thyroxine concentrations as low as 5 X lo-’ M can potentiate hemolysis. Involvement of radicals. In order to determine the contribution of various free radicals to the thyroid hormone-peroxide induced hemolysis, two experiments were performed. The participation of Oi was assayed by the inclusion of SOD (28 U/ml) in the incubation medium. During incubations of up to 5 h at 37°C with the peroxide generating system and 1 x lo-:’ M thyrox-
TABLE DIFFERENTIAL
I OF OLD
SENSITIVITY
CELLS TO THYROXINE-PEROXIDE HEMOLYSIS AND PROTECTION
G-B-PD Activity (% unfractionated blood
AND
YOUNG
INDUCED BY GLUCOSE
Lysis
(%I
H,O,
H,O, + T,
-___ H,O, + T, + glucase
sample)
Unfractionated Young Old
100 111 46
6 4 48
49 45 78
12
7 60
OF
HUMAN
RED
465
CELLS
100 1 80 . 52 ;c 6 60. * ; z 40
/I/p ._:;/
20
i
0
,510
.-- r A--__-_-___----
_<*-
__--
o _____ - ----50 L-T, x10”M
--.
--o 100
FIG. 1. Thyroxine concentration curve. The amount of hemolysis obtained at varying thyroxine concentrations was determined in the presence of the peroxide generating system after 3 (0) and 5 (0) h of incubation at 37°C. The dashed lines (- --) indicate the presence of 100 mg% glucose.
ine, there was no diminution of their hemolytic effect. The possible involvement of the hydroxy radical (OH’) was determined by the addition of OR scavengers (i.e., ethanol, mannitol, glucose). Figure 2 illustrates the inhibitory effect of ethanol (5.8 mM), mannito1 (5.5 mM), and glucose (5.5 mM) on thyroxine-peroxide induced hemolysis. The results suggest that the OH may be involved in these cytotoxic events and that the protective effect of glucose may involve its capacity to scavenge hydroxy radicals as well as its metabolic role in the detoxification of H,O,. Prelytic lipid peroxidation. The formation of MA in the presence of the thyroxine-peroxide hemolytic system was measured in order to determine the possible role of lipid peroxidation in the hemolytic process. The experiment was performed in the presence of 1 X lo-” M thyroxine or 1 X lo-” M triiodothyronine. All determinations were made prior to the onset of measurable hemolysis in order to assure the prelytic nature of MA production. The results are shown in Fig. 3. It is clear that the thyroid hormones increase the amount of MA formation during the course of these incubations.
466
WALLS,
KUMAR
AND
HOCHSTEIN
the hemolytic system. Although there was no detectable lysis in these experiments, some methemoglobin formation was observed. It is of interest that the amount of methemoglobin formed was somewhat less in the presence of thyroxine. Filtration of’ red cells. After 1 h of incubation at 37°C in the presence of the peroxide generating system with and without 1 x lo-” M thyroxine, the cells were filtered through 5-pm pore membranes. The results are shown in Table III. Each sample was assayed in triplicate. It can be seen that the cells exposed to the peroxide and thyroxine were retained by the filters to a greater extent than those exposed only to the peroxide generating system. Thyroxine alone had no effect on the filtration of red cells. 2
4
6
hours FIG. 2. The effect of hydroxy radical scavengers on thyroxine/peroxide induced hemolysis. The initial L-T, concentration was 1 x lo-” M. PCS represents the peroxide generating system. Glucose and mannitol were 5.5 mM; ethanol was 5.8 rn~ final. Each point represents the average of closely agreeing duplicate determinations.
DISCUSSION
The biological effects of peroxide and oxygen radicals have received considerable TABLE K+ LEAK GENERATING
IN
THE
(H.,O,)
SYSTEM
Additions
II
PRESENCE
OF PEROXIDE 1 x lo-”
AND
Micrograms
of K- per milliliter SD
0 min None H,O, H,O, L-T,
1.67 2.17 2.13 1.57
+ L-T,
EFFECT
FIG. 3. Prelytic lipid peroxidation. Final concentrations of T:, and T, were lo- i M. PGS signifies the peroxide generating system. Each point represents the average of closely agreeing duplicate determinations.
Prelytic K+ loss. Table II illustrates the effect of thyroxine and peroxide on the K+ leak from red cells. After 60 min of incubation at 37”C, there was an increase in the amount of K+ lost by the cells exposed to
Background buffer IiltrationO time (B,)
3.17 2 0.55 2.90 2 0.61 4.80 -t 0.26 3.20 i 0.62
III
OF PEROXIDE
ERYTHROCYTE
lr
60 min
-+ 0.25 t 0.55 rt_ 0.49 i 0.12
TABLE THE
M L-T,
AND
THYROXINE
ON
FILTERABILITY ___-
Sample filtration” time (S,)
S,IB,
H,O, 8.7 9.2 8.9
10.8 11.2 12.0
8.3 8.7 9.3
14.7 22.0 23.6
H,Os
a In seconds.
1.24 1.22 1.35
1.27 I? .07
1.77 2.53 2.54
2.28 I .44
+ T,
THYROXINE
AND
AGING
attention in recent years. These oxidants may be generated through the activity of flavin enzymes, during irradiation, and by the autoxidation of drugs (9, 10). Physiological processes such as phagocytosis by leukocytes are accompanied by the production of peroxide and other oxygen radicals (11). The ability of cells to control the concentrations of these oxidants is essential to their continued survival. In red blood cells, the capacity to detoxify hydrogen peroxide involves the enzyme glutathione peroxidase whose activity is coupled to that of G-6PD through glutathione reductase (2, 12). Thus, the activity of G-6-PD, which controls the entry of reducing equivalents into this system, is the regulating factor in the detoxification of hydrogen peroxide. Low levels of activity or absence of this enzyme, as in G-6-PD deficiency, deprives cells of their capacity to destroy peroxide. This results in the accumulation of oxidants within the cell and damage manifested by oxidation of glutathione, the formation of methemoglobin, the peroxidation of membrane lipids, and eventual hemolysis. In previous experiments, we have demonstrated that thyroxine potentiates the hemolytic effects of peroxide (4). Peroxidase catalyzed hemolysis utilizing thyroxine as a halogen source also has been observed (13). The hemolysis described in the current studies apparently results from a complex series of events. The formation of methemoglobin, increased K+ leak, oxidant injury catalyzed by OH radicals, and peroxidation of membrane lipids are described in this paper. Although not included in this report, the iodination of membrane components may also be involved in the cytotoxic events (13). Although the responses to thyroxine, demonstrated in this study, were at concentrations above the physiological level, two factors must be considered. First, the thyroxine concentration in vitro is continually decreasing whereas in vim homeostatic mechanisms maintain hormone levels over long periods of time. Second, the limited solubility of thyroxine near neutral pH makes it certain that the amount of hormone available for reaction in these experiments is much less than that added.
OF
HUMAN
RED
467
CELLS
It may be of special significance that cells exposed to thyroxine and peroxide also exhibit a decreased filterability (Table III). Such a prelytic event may reflect alterations in surface to volume ratios and a consequent decrease in deformability (14). The mechanisms of splenic recognition of senescent erythrocytes have not been fully elucidated. It may well be that peroxide produced by splenic phagocytes, in the presence of circulating thyroxine, during acts to “condition” erythrocytes their passage through the spleen so that eventual entrapment of rigid, senescent cells takes place. Such entrapment may be enhanced by a variety of chemical alterations and immunological events which lead to erythrophagocytosis. However, the hypersensitivity of the old cells to peroxide and thyroxine illustrated by these studies, and the lack of protection afforded by glucose, substantiates the view that thyroid hormone may be a major factor in the sequestration of red cells in narrow splenic channels. It should also be noted that the activity of G-6-PD is known to decrease with erythrocyte aging (15, 16). This decrease is associated with a decrease in GSH concentration (17) and increased methemoglobin formation (18) in older cells. The decreased life-span of G-6-PD deficient cells (19) and the purported shortened survival time of red cells from hyperthyroid individuals (5) support an hypothesis that peroxide and thyroxine are intimately involved in the aging of red cells. ACKNOWLEDGMENT This work (HD 08159).
was supported
in part
by an NIH
Grant
REFERENCES 1. BUNN, 2. COHEN, 134,
H. F. (1972) G.,
AND
Semin.
HOCHSTEIN,
Henatol. 9, 3. P. (1961) Science
1756.
BEUTLER, E. (1969) Pharnz. Rruieu, 21, 73. WALLS, R., AND HOCHSTEIN, P. (1974) Lifr 15, 1757. 5. MCCLELLAN, J. E., DONEGAN, C., THORUP,
3. 4.
Sci.
0. A., AND LEAVELL, B. S. (1958) J. Lab. Clin. Med. 51, 91. 6. BEUTLER, E. (1971) Red Cell Metabolism, p, 62, Grune and Stratton, New York/London. 7. PIOMELLI, S., LURINSKY, G., AND WASSERMAN,
468
WALLS, L. R. (1967) J. Lab.
8. MCCORD,
J.,
AND
Clin.
FRIDOVICH,
Chem. 144, 6049. 9. COHEN, G., AND HEIKKILA, Chem. 249, 2447.
KUMAR
Med. 69, 659. I. (1969) J. Biol. R. E. (1974)
J. Biol.
G., AND HOCHSTEIN, P. (1964) Biochem3, 895. 11. PAUL, B., AND SBARRA, A. J. (1968) Biochim. Biophys. Acta 156, 168. 12. COHEN G., AND HOCHSTEIN, P. (1963)Biochemk try 2, 1420. 13. KLEBANOFF, S. J., AND CLARK, R. A. (1975) Blood 45, 699. 10.
COHEN,
istry
AND
HOCHSTEIN HARADIN, (1969)
15. 16.
MARKS, P. A. (1957) J. Clin. Inuest. 36, 913. RAMOT, B., BROK-SIMONI, R., AND BEN-BASSAT, I. (1969) Ann. N.Y. Acad. Sci. 165, 400.
17. RIGAS, Clin.
A. R., Traufusion.
WEED, R. I., AND 9, 229.
14.
D. A.,
REED,
C. F.
AND KOLER, R. D. (1961) J. Lab. Med. 58, 417. 18. KEITT, A. S., SMITH, T. W., AND JANDL, J. H. (1966) New Eng. J. Med. 275, 397. 19. BERNINI, L., LATTE, B., SINISCALCO, M., PIOMELLI, S., SPADA, U., ADINOLFI, M., AND MOLLISON, P. L. (1964)Brit. J. Haematol. 10, 171.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Aging Differential
Sensitivity
174, 463-468
of Human
(1976)
Erythrocytes
of Young and Old Erythrocytes to Hemolysis Peroxide in the Presence of Thyroxine
RICHARD
WALLS,
Department
ofPharmacology,
K. SREE
KUMAR,
University Los Angeles, Received
AND
ofsouthern California
November
PAUL
California 90033
Induced
by
HOCHSTEIN School
ofMedicine,
10, 1975
Human erythrocytes were separated according to cell age using albumin density gradients. In the presence of glucose (100 mg%), young cells were able to effectively protect themselves against thyroxine-peroxide induced hemolysis; old cells exhibited less protection. Hemolysis in heterogeneous populations is preceded by lipid peroxidation, K+ leak and decreased filtrability of the cells. Hydroxy radical scavengers partially inhibited hemolysis while superoxide dismutase had no effect. It is postulated that the differential sensitivity of young and old erythrocytes to thyroxine-peroxide induced metabolic and morphological alterations may play a role in the recognition and removal of senescent cells from the circulation.
Human erythrocytes have a finite life span of about 120 days. Their aging in vivo is accompanied by a decrease in the activity of enzymes such as glucose-6-phosphate dehydrogenase (G-6-PD)’ as well as by alterations in other cellular components (1). G-6-PD also plays a cardinal role in the detoxification of hydrogen peroxide (2). It is well established that red cells with a genetic deficiency of this enzyme undergo oxidative alterations when exposed to peroxide (3). We recently reported that the hemolysis of such cells in the presence of hydrogen peroxide was markedly enhanced by thyroid hormone (4). We suggested that thyroxine is a potentiating factor in the anemia associated with oxidative stress in G-6-PD-deficient individuals and that the toxicity of autoxidizable drugs is a consequence of both the formation of hydrogen peroxide and the availability of thyroid hormones. This concept raises the question of ’ Abbreviations used: G-6.PD, glucose-6-phosphate dehydrogenase: L-T,, L-thyroxine; BSG, buffered-saline-glucose; MA, malonaldehyde; TBA, Z-thiobarbituric acid; SOD, superoxide dismutase.
METHODS Hemolysis volunteers 463
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
whether thyroxine might also participate in the recognition and destruction of normal erythrocytes during the aging process. In this connection, McClellan et aZ. (5) have reported that the survival time of the erythrocytes of hyperthyroidic individuals is shortened, whereas the transfusion of these cells to euthyroid persons results in their normal survival. The experiments described in this paper were undertaken to determine whether or not older normal cells, which have decreased G-6-PD activity, exhibit an increased sensitivity to the cytotoxic effects initiated by peroxides in the presence of thyroxine. Our results demonstrate that, in fact, the response of older cells to these agents is similar to that of G-6-PD delicient cells and support the concept that thyroxine may be involved in the aging process. We also describe the nature of some of the prelytic events which may be relevant to the entrapment of senescent cells in the circulation. experiments. was collected
Blood from healthy, adult either into heparinized,
464
P
WALLS,
KUMAR
disposable syringes or defibrinized with glass beads. The cells were washed three times with 0.9% NaCl0.1% human albumin, and the final hematocrit was adjusted to 30%. The cells were utilized within 24 h. Erythrocytes (1% hematocrit) were exposed to a peroxide generating system (1.6 x lo-’ U/ml xanthine oxidase and 1.67 x 10m3 M hypoxanthine) with and without the inclusion of L-thyroxine (L-T,) in a final 3.0-ml reaction volume. A modified KrebsRinger phosphate buffer, pH 7.0, (48 mM K-PO,, pH 7.0; 73 mu NaCl; 5 mM KCl; 1.2 InM MgSO,.7 H,O; 0.9 mM CaC1.2 H,O) was used as the incubation medium. When glucose was included in the reaction mixture, the final concentration was 100 mg% (5.5 mM). L-Thyroxine was prepared in 0.05 N NaOH. All incubations were at 37°C with continuous shaking in lo- or 5-ml conical flasks. One-milliliter samples were withdrawn from incubation flasks at desired time intervals and centrifuged at 1OOOg for 5 min. Supernatant (0.65 ml) was then removed and mixed with 1.3 ml of Drabkin’s reagent (100 mg of NaCN and 300 mg of K,,Fe(CN),, in 1000 ml of H,O) (6), and the extent of hemolysis was determined at 540 nm using a Gilford Spectrophotometer. One hundred percent hemolysis was determined by adding 0.1 ml of 30% erythrocytes to 2.9 ml of glass-distilled H,O. This sample was then assayed for hemolysis as described above. Separation of old and young cells. The technique of Piomelli et al. (7) for rabbit cell fractionation was modified to suit human erythrocytes. Buffered-saline-glucose (BSG; 8.12 g NaCl, 1.22 g Na,HPO,, 0.219 g NaH,P0,,2H,O, 2.0 g glucose, made up to 1000 ml glass-distilled H,O) was used to wash the red cells obtained from defibrinated blood. The cells were then packed by centrifugation at 1OOOg for 5 min. A stock solution of 46 gYo albumin was prepared in water. The stock albumin solution was diluted with BSG to form three solutions with specific gravities of 1.087, 1.091, and 1.095. The gradient was prepared by layering equal volumes (ca. 3.0 ml) of each of the albumin solutions carefully into 12-m], round bottom, polypropylene tubes, starting with the most dense working up to the least. Packed cells (1.5-2.0 ml) were added to the top of the gradient. The tubes were then centrifuged at 2-4°C for 60 min at 39,000 rpm using an SB283 rotor in an IEC, Model B60, ultracentrifuge. The cells separated into four bands, including the bottom sediment and were aspirated into tubes from the top of the gradient by applying a small negative pressure. The cells were washed in saline and adjusted to a final 30% hematocrit with saline-albumin. The lowest band (fraction 4) corresponded to the older, most dense cells, whereas the top band (fraction 1) represented the younger cells. Glucose-&phosphate dehydrogenase (G-6-PD) activity was determined according to the method of Beutler (6).
AND
HOCHSTEIN
TBA reactive material. Malonaldehyde (MA) was determined in l.O-ml suspensions of 1.0% red cells. The l.O-ml sample was added to 0.1 ml of 50% trichloracetic acid and centrifuged at 1OOOg for 5 min to form a hard-packed pellet. The supernatant was removed and added to 0.5 ml 0.75% 2-thiobarbituric acid (TBA). Samples were boiled for 20 min and cooled to room temperature. Scans were made between 600 and 450 nm to determine the spectra of the chromogens. The MA-TBA chromogen has a maximum at 532 nm. Because of varying differences in background absorption, the MA formed was calculated by the difference in the readings obtained between 532 and 580 nm. K+ leak studies. The reaction mixture for hemolysis studies was used except that the buffer was replaced by 0.1 M Na-PO,, pH 7.0. After indicated time periods, the samples were centrifuged and 1.0 ml of the supernatant was removed. Glass-distilled deionized water (1.0 ml) was then added to the supernatant sample and the K+ was determined using a Perkin-Elmer Atomic Absorption Spectrophotometer, Model 290. All K+ determinations were made prior to the onset of hemolysis in order to assure the K’ loss as a prelytic event. Filtration studies. These studies were conducted using a Millipore filtration apparatus with 5-pm pore, 25-mm diameter Nucleopore membranes. The membranes were soaked in double-distilled water and sonicated prior to use. Cells were incubated in the presence of the peroxide generating system with or without 1 x lo-” M thyroxine for 1 h at 37°C. Filtration times were determined on 3.0-ml samples by recording the flow time of 1.0 ml after an initial 0.5 ml passed through the membrane. Xanthine oxidase (Sigma), hypoxanthine (Sigma), L-thyroxine (Nutritional Biochemicals), triiodothyronine (Calbiochem), and bovine albumin, Fraction V (Pentex, Miles Laboratories) were purchased. Superoxide dismutase (SOD) was prepared according to the method of McCord and Fridovich (8). One unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of cytochrome c by 50% at an initial rate of 0.5 ODI min. RESULTS
Differential sensitivity of old and young cells to glucose protection against hemolysis. The G-6-PD activity was determined
on the various cell fractions prepared on albumin gradients and compared to the activity of an unfractionated blood sample to assure age differences. The cells were then exposed to the peroxide generating system with or without L-thyroxine (lo-;’ M) and with or without 100 mg% glucose.
THYROXINE
AND
AGING
The amount of hemolysis was determined after 21/z h incubation at 37°C. The results are shown in Table I. The data, with the exception of the G-6-PD activity, are expressed as the percentage of hemolysis obtained. It is clear that in the absence of glucose, all cell populations are susceptible to hemolysis when exposed to the peroxide generating system and L-T,. In the presence of glucose, the younger cells are more able to protect themselves against hemolysis; whereas the older cells exhibited less protection. In the presence of glucose, unfractionated and young cells exhibited 75 to 85% protection against hemolysis, however the older cells exhibited only 25% protection. Thyroxine concentrations. The effect of various thyroxine concentrations and the peroxide generating system on a normal, heterogeneous population of cells is illustrated in Fig. 1. The protective effect of glucose (100 mg%) is also demonstrated. Hemolysis was assayed after 3 and 5 h incubation at 37°C. The results indicate that under the conditions of these experiments, thyroxine concentrations as low as 5 X lo-’ M can potentiate hemolysis. Involvement of radicals. In order to determine the contribution of various free radicals to the thyroid hormone-peroxide induced hemolysis, two experiments were performed. The participation of Oi was assayed by the inclusion of SOD (28 U/ml) in the incubation medium. During incubations of up to 5 h at 37°C with the peroxide generating system and 1 x lo-:’ M thyrox-
TABLE DIFFERENTIAL
I OF OLD
SENSITIVITY
CELLS TO THYROXINE-PEROXIDE HEMOLYSIS AND PROTECTION
G-B-PD Activity (% unfractionated blood
AND
YOUNG
INDUCED BY GLUCOSE
Lysis
(%I
H,O,
H,O, + T,
-___ H,O, + T, + glucase
sample)
Unfractionated Young Old
100 111 46
6 4 48
49 45 78
12
7 60
OF
HUMAN
RED
465
CELLS
100 1 80 . 52 ;c 6 60. * ; z 40
/I/p ._:;/
20
i
0
,510
.-- r A--__-_-___----
_<*-
__--
o _____ - ----50 L-T, x10”M
--.
--o 100
FIG. 1. Thyroxine concentration curve. The amount of hemolysis obtained at varying thyroxine concentrations was determined in the presence of the peroxide generating system after 3 (0) and 5 (0) h of incubation at 37°C. The dashed lines (- --) indicate the presence of 100 mg% glucose.
ine, there was no diminution of their hemolytic effect. The possible involvement of the hydroxy radical (OH’) was determined by the addition of OR scavengers (i.e., ethanol, mannitol, glucose). Figure 2 illustrates the inhibitory effect of ethanol (5.8 mM), mannito1 (5.5 mM), and glucose (5.5 mM) on thyroxine-peroxide induced hemolysis. The results suggest that the OH may be involved in these cytotoxic events and that the protective effect of glucose may involve its capacity to scavenge hydroxy radicals as well as its metabolic role in the detoxification of H,O,. Prelytic lipid peroxidation. The formation of MA in the presence of the thyroxine-peroxide hemolytic system was measured in order to determine the possible role of lipid peroxidation in the hemolytic process. The experiment was performed in the presence of 1 X lo-” M thyroxine or 1 X lo-” M triiodothyronine. All determinations were made prior to the onset of measurable hemolysis in order to assure the prelytic nature of MA production. The results are shown in Fig. 3. It is clear that the thyroid hormones increase the amount of MA formation during the course of these incubations.
466
WALLS,
KUMAR
AND
HOCHSTEIN
the hemolytic system. Although there was no detectable lysis in these experiments, some methemoglobin formation was observed. It is of interest that the amount of methemoglobin formed was somewhat less in the presence of thyroxine. Filtration of’ red cells. After 1 h of incubation at 37°C in the presence of the peroxide generating system with and without 1 x lo-” M thyroxine, the cells were filtered through 5-pm pore membranes. The results are shown in Table III. Each sample was assayed in triplicate. It can be seen that the cells exposed to the peroxide and thyroxine were retained by the filters to a greater extent than those exposed only to the peroxide generating system. Thyroxine alone had no effect on the filtration of red cells. 2
4
6
hours FIG. 2. The effect of hydroxy radical scavengers on thyroxine/peroxide induced hemolysis. The initial L-T, concentration was 1 x lo-” M. PCS represents the peroxide generating system. Glucose and mannitol were 5.5 mM; ethanol was 5.8 rn~ final. Each point represents the average of closely agreeing duplicate determinations.
DISCUSSION
The biological effects of peroxide and oxygen radicals have received considerable TABLE K+ LEAK GENERATING
IN
THE
(H.,O,)
SYSTEM
Additions
II
PRESENCE
OF PEROXIDE 1 x lo-”
AND
Micrograms
of K- per milliliter SD
0 min None H,O, H,O, L-T,
1.67 2.17 2.13 1.57
+ L-T,
EFFECT
FIG. 3. Prelytic lipid peroxidation. Final concentrations of T:, and T, were lo- i M. PGS signifies the peroxide generating system. Each point represents the average of closely agreeing duplicate determinations.
Prelytic K+ loss. Table II illustrates the effect of thyroxine and peroxide on the K+ leak from red cells. After 60 min of incubation at 37”C, there was an increase in the amount of K+ lost by the cells exposed to
Background buffer IiltrationO time (B,)
3.17 2 0.55 2.90 2 0.61 4.80 -t 0.26 3.20 i 0.62
III
OF PEROXIDE
ERYTHROCYTE
lr
60 min
-+ 0.25 t 0.55 rt_ 0.49 i 0.12
TABLE THE
M L-T,
AND
THYROXINE
ON
FILTERABILITY ___-
Sample filtration” time (S,)
S,IB,
H,O, 8.7 9.2 8.9
10.8 11.2 12.0
8.3 8.7 9.3
14.7 22.0 23.6
H,Os
a In seconds.
1.24 1.22 1.35
1.27 I? .07
1.77 2.53 2.54
2.28 I .44
+ T,
THYROXINE
AND
AGING
attention in recent years. These oxidants may be generated through the activity of flavin enzymes, during irradiation, and by the autoxidation of drugs (9, 10). Physiological processes such as phagocytosis by leukocytes are accompanied by the production of peroxide and other oxygen radicals (11). The ability of cells to control the concentrations of these oxidants is essential to their continued survival. In red blood cells, the capacity to detoxify hydrogen peroxide involves the enzyme glutathione peroxidase whose activity is coupled to that of G-6PD through glutathione reductase (2, 12). Thus, the activity of G-6-PD, which controls the entry of reducing equivalents into this system, is the regulating factor in the detoxification of hydrogen peroxide. Low levels of activity or absence of this enzyme, as in G-6-PD deficiency, deprives cells of their capacity to destroy peroxide. This results in the accumulation of oxidants within the cell and damage manifested by oxidation of glutathione, the formation of methemoglobin, the peroxidation of membrane lipids, and eventual hemolysis. In previous experiments, we have demonstrated that thyroxine potentiates the hemolytic effects of peroxide (4). Peroxidase catalyzed hemolysis utilizing thyroxine as a halogen source also has been observed (13). The hemolysis described in the current studies apparently results from a complex series of events. The formation of methemoglobin, increased K+ leak, oxidant injury catalyzed by OH radicals, and peroxidation of membrane lipids are described in this paper. Although not included in this report, the iodination of membrane components may also be involved in the cytotoxic events (13). Although the responses to thyroxine, demonstrated in this study, were at concentrations above the physiological level, two factors must be considered. First, the thyroxine concentration in vitro is continually decreasing whereas in vim homeostatic mechanisms maintain hormone levels over long periods of time. Second, the limited solubility of thyroxine near neutral pH makes it certain that the amount of hormone available for reaction in these experiments is much less than that added.
OF
HUMAN
RED
467
CELLS
It may be of special significance that cells exposed to thyroxine and peroxide also exhibit a decreased filterability (Table III). Such a prelytic event may reflect alterations in surface to volume ratios and a consequent decrease in deformability (14). The mechanisms of splenic recognition of senescent erythrocytes have not been fully elucidated. It may well be that peroxide produced by splenic phagocytes, in the presence of circulating thyroxine, during acts to “condition” erythrocytes their passage through the spleen so that eventual entrapment of rigid, senescent cells takes place. Such entrapment may be enhanced by a variety of chemical alterations and immunological events which lead to erythrophagocytosis. However, the hypersensitivity of the old cells to peroxide and thyroxine illustrated by these studies, and the lack of protection afforded by glucose, substantiates the view that thyroid hormone may be a major factor in the sequestration of red cells in narrow splenic channels. It should also be noted that the activity of G-6-PD is known to decrease with erythrocyte aging (15, 16). This decrease is associated with a decrease in GSH concentration (17) and increased methemoglobin formation (18) in older cells. The decreased life-span of G-6-PD deficient cells (19) and the purported shortened survival time of red cells from hyperthyroid individuals (5) support an hypothesis that peroxide and thyroxine are intimately involved in the aging of red cells. ACKNOWLEDGMENT This work (HD 08159).
was supported
in part
by an NIH
Grant
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