Vol. XXIV, No. 5 Printed in USA.
JOURNAL OF THE AMERICAN GERIATRICS SOCIETY Copyright 0 1976 by the American Geriatrics Society
Free Radical Theory of Aging: Inhibition of Amyloidosis in Mice by Antioxidants; Possible Mechanism* DENHAM HARMAN, MD, PhD**, DENNIS E. EDDY, PhD and JAMES NOFFSINGER, PhD Departments of Biochemistry and Medicine, University of Nebraska College of Medicine, Omaha, Nebraska
ABSTRACT: The antioxidants, a-tocopherol acetate and a quinolone derivative (Santoquin), inhibited the development of amyloidosis when added to the diet of casein-injected C3HeB/FeJ male mice. Santoquin, and to a lesser extent butylated hydroxytoluene (BHT), also depressed the appearance of a plasma protein fraction in these mice; the effect of a-tocopherol was not determined. Consideration of the current knowledge of amyloid, in the light of these antioxidant studies, prompted the following hypothesis. Amyloidosis is largely the result of an enhanced rate of oxidative degradation of a connectivetissue glycoprotein(s) coupled with oxidative/enzymatic changes in the plasma, both of the tissuederived substances and of immunoglobulins, to form the amyloid fibril protein subunits (ALand AA) which subsequently aggregate to form the amyloid fibrils.
The incidence of amyloidosis increases with age (1, 2). The reason for this association, as well as the pathogenesis of amyloidosis, is unknown. Amyloid is associated with a large number of seemingly unrelated diseases (2), both in man and in many species of animals. It can be induced in animals by a wide variety of methods (2, 3). Amyloid material from different sources has essentially the same appearance (2, 3). Under the light-microscope, it appears as an amorphous, hyaline, extracellular material, readily detected by its characteristic staining properties. In the electron-microscope, morphologically unique fibrils appear as the major constituent of amyloid. An observation made during the course of a study of the effect of antioxidants on the lifespan of male LAF, mice, based on the possibility that endogenous free radical reactions contribute to the degradation of biologic systems (4),may aid in shedding light on both the etiology of amyloidosis and its association with age. * The authors gratefully acknowledge the financial assistance of the late Mrs. Adah Millard of Omaha, Nebraska and of Mr. Myron Pfeifer of Concord, Tennessee. * * Address for correspondence: Denham Harman, MD, Department of Biochemistry, College of Medicine, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, NB 68105.
LAF, mice are prone to develop amyloidosis (5). Marked deposits of amyloid are found in the spleen, liver and kidneys of many mice of this strain a t death. The incidence of amyloidosis at death is diet-dependent. Mice fed a commercial pelleted diet have about a 25 percent incidence (5) of amyloidosis, but an approximately 65 percent incidence (6) if given a semisynthetic diet containing 2 0 % ~ (percent by weight) casein as the sole source of protein. The average lifespan of male LAF, mice fed a 2 0 % ~casein diet was markedly increased by adding to the diet 0.25-0.50%w of butylated ~ 1,2-dihyhydroxytoluene (BHT) (6) or 0 . 2 5 % of dro-6-ethoxy-2,2,4-trimethyquinoline(7) (Santoquin, Ethoxyquin-Monsanto Chemical Company, St. Louis, Missouri); another antioxidant, a-tocopherol acetate, had little if any effect (7). The increase in lifespan produced by the two antioxidants was paralleled by a decrease in the incidence of amyloidosis. This observation suggested that the beneficial effect of the antioxidants on average lifespan was due, at least in part, to inhibition of a free radical reaction(s) involved in amyloidosis. The purpose of the present study was to determine if casein-induced mouse amyloidosis would
203
VOl. XXIV
HARMAN, EDDY AND NOFFSINGER
distilled water for 48 hours; the water was changed frequently. The solution was then transferred to an ultra filtration apparatus (Amicon Corp., Lexington, Massachusetts), and concentrated to 25 ml by means of an Amicon Type XMlOOA membrane. The final 10% casein solution was stored in the cold room until use; storage time did not exceed 10 days.
also be inhibited by the addition of an antioxidant to the diet. Santoquin, BHT and a-tocopherol acetate were evaluated. METHODS AND RESULTS Two casein-induced amyloid experiments were carried out in sequence on C3HeBPeJ male mice. The mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) shortly after weaning, and then caged 10 per cage (stainless steel, 12x8 x 5 inches). They were maintained in an airconditioned room a t 76-78' F a t a humidity of 50-60 percent. The cages were changed twice a week. The bedding consisted of sterilized shredded corn cobs (San-i-Cel, Paxton Processing Company, Paxton, Illinois). Light was from overhead fluorescent lamps automatically controlled to provide light from 6:OO a.m. to 8:OO p.m. The mice were fed a commercial pelleted mouse chow (Rockland; Teklad, Inc., Monmouth, 11linois) from the time of arrival until they were about 40 days of age, when the experiments were started. Amyloidosis was induced by the subcutaneous injection of 0.3 ml of a 1 0 % (percent ~ by weight) aqueous solution of casein (8) 5 days per week, starting around 75 days of age. The casein solution was prepared by dissolving 2.5 gm of vitaminfree test casein (Teklad Mills, Madison, Wisconsin) in 50 ml of 0.25% NaOH. The casein solution was next dialyzed a t 4°C (cold room) against
Experiment A-Effect
50
57 63 71 7Sb
86 92 99 1 W 113 120 126 134 141 148 154
No.
Avg. Wt.
30 30 30 30 30 30 30 29 29 28 24 20 16 12 7 5
18.4 21.3 22.4 23.4 24.2 24.7 24.7 26.0 26.6 27.4 27.9 27.8 28.6 27.7 28.1 27.8
0
In the first experiment, 120 mice were divided into four dietary groups of 30 each, a t age 43 days. They were fed a powdered diet (Rockland) to which was added 0 . 2 5 % ~of BHT, or 0 . 2 5 % ~of Santoquin, or nothing (the pair-fed controls for the antioxidant groups). A t 78 days of age, subcutaneous injection of 1 0 % casein ~ solution was started (0.3 ml per mouse five days per week). Food consumption and body weight data are presented in Table 1. There were no significant differences between the four groups except for a modest decrease in food consumption in the BHT group beginning a t age 134 days. Data on the 7 mice that died after the start of casein injections are shown in Table 2. Starting a t 106 days of age, a t short intervals 2 mice from each group underwent autopsy until the end of the experiment a t age 155 days. This autopsy schedule was prompted by an initial study employing 50 mice; splenic sections obtained before 120 days of age were free of amyloid whereas after
TABLE 1 of Antioxidants on Casein-Induced Amyloidosk in Male C3HeBIFd Mice: Average Body Weight and Food Consumption Control Id
BHT 0.25%~
43
Experiment A
-
Control T
Santoquin 0.25%~
Food Intake'
No.
Avg. Wt.
Food Intake
13.7 20.5 19.5 19.5 17.6 17.4 17.9 20.3 20.3 21.0 19.6 20.3 18.9 17.5 18.2 14.0
30 30 30 30 30 30 29 29 29 29 25 21 17 13 9 7 3
19.0 22.7 23.7 24.5 25.0 25.4 26.2 26.8 27.4 28.0 28.2 28.5 29.1 29.5 30.1 30.0 27.7
18.4 20.8 19.4 20.3 19.0 18.7 19.0 20.2 21.2 22.4 21.7 22.3 24.5 24.5 23.1 23.1
No.
30 30 30 30 30 30 30 30 29
29 25 21 17 13 9 7 3
Avg. Wt.
18.4 22.1 23.0 23.8 24.6 24.9 25.5 26.0 26.4 27.2 27.4 27.2 27.8 28.1 27.8 26.7 27.7
Food Intake
17.4 21.2 20.5 20.9 17.4 17.9 19.6
21.6 19.6 21.0 21.0 22.3 21.7 21.0 19.5 21.0
Avg.
No.
Wt.
30 30 30 30 30 30 30 29 28
18.0 21.6 22.9 24.0 24.4 24.9 25.7 26.2 26.9 27.1 27.8 28.1 28.3 27.8 27.0 27.0 25.0
28 24 20 16 12 8 6 2
Control 3'
Food Intake
18.7 20.6 18.6 18.5 17.0 17.9 18.0 19.9
20.3 22.2 20.3 21.7 22.4 18.9 18.6 16.8
No. 10 10 10 10 10 10 10 10 10 10 8
7 6 5 4 4 4
Avg. Wt.
17.9 21.6 23.1 24.2 24.2 24.6 25.8 26.0 26.6 27.3 27.9 28.1 28.1 28.2 28.2 28.2 28.5
Food Intake
18.2 22.5 19.9 18.2 17.2 18.4 18.6 20.6 19.1 21.3 18.9
21.0 23.8 25.2 22.4 28.0
Foodhnouselweek. Casein iniections started. At age 106 days.4 animals were killed per week in the first 4 groups and 1 animal per week in Control 3 (water-injected grcup), slides made, and degree of amyloidosis noted. Pair-fed control for the 0.25%~BHT group. 'Pair-fed control for the 0.25%~Santoquin group. ' Pair-fed with the Santoquin group, injected with distilled water, a
204
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS TABLE 2 Experiment A-Effect of Antioxidants on Casein-Induced Amyloidosis in Male C3HeBIFeJ Mice: Data on Mice Dying after Casein Injections Were Started at 78 Days of Age Santoquin
Age (days)
Cage
Control 1
No. Dead
85 92 98
99
8
BHT
Cage
No. Dead
4
1‘
Cage
No. Dead,
Cage
No. Dead
2
1
11 11
1 1
3 1
1 (3+P 1 (2+P
1.
134 149 a
Control 2
No autopsy. Spleen amyloid, graded on basis of 0-4+
age 150 days, when autopsies were resumed, all splenic sections were about the same (3.5+ for amyloid). Sections of spleen and liver were stained with hemotoxylin and eosin (H & E). The spleen sections were graded on three separate occasions for amyloid (on the basis of 0-4+) by the same individual and the average of the three gradings recorded; the observer did not know the identity of the slides. Initially the liver sections were also graded for amyloidosis; the gradings were stopped as they were parallel to, but less than, the splenic values. The splenic gradings are shown in Table 3. The incidence of splenic amyloidosis in the BHT-treated group was not significantly different from that in its pair-fed control group, although some BHT mice in the latter part of the experiment were free of amyloid whereas none of the controls were after age 134 days. In contrast, the Santoquin group had significantly less (P < 0.05; Mann-Whitney U test) splenic amyloidosis than the control group. A blood sample was obtained from each mouse at autopsy and the plasma (EDTA) subjected to cellulose acetate electrophoresis (9); the strips were stained for glycoprotein (9) and the relative amounts of each fraction measured by densitometry. The densitometer tracings were all similar except for a component (a-glycoprotein “X”) in the a,-region indicated by an arrow in Figure 1. This component, seen first at 109 days, was observed with increasing frequency as the number of casein injections increased, but tended to be less in the Santoquin and BHT groups. The a,-glycoprotein “X” peak was also observed in the plasma of water-injected mice (pair-fed with the Santoquin group), beginning at age 130 days. The proportion of mice in each group with the plasma glycoprotein “X” peak was: BHT, 34 percent (10/29); BHT control, 83 percent (24/29); Santoquin, 17 percent (5/29); Santoquin control,
64 percent (16/25); and sterile-water injected controls, 60 percent (6/10). In Table 3 the area of the small peak in the a,-glycoprotein region (see Figure 1) is listed in the section below the corresponding splenic gradings. A few plasma (EDTA) samples were available for polyacrylamide gel electrophoresis (10); the gels were stained for glycoprotein (11). A band in the a,-globulin region was decreased (indicated by the arrow in Figure 2) in the plasma of several mice that had received Santoquin in their diet; this decrease was not observed in the one sample of plasma run from the BHT group.
Experiment B Experiment B was carried out to confirm the Santoquin results of Experiment A and to evaluate the effect of a-tocopherol acetate on caseininduced amyloidosis. For the second study, 180 mice were divided into four groups, 40 in each of two treated groups and 50 in each of two control groups, at age 35 days, and given a powdered diet to which was added 0 . 2 5 % Santoquin, ~ or 0 . 2 5 % ~ a-tocopherol acetate, or nothing (the pair-fed controls for the two antioxidant groups). At 77 days of age, subcutaneous injection of casein solution was begun, as described in Experiment A. Weekly food consumption and body weight data are shown in Table 4. No significant differences were noted. Data on the 42 mice that died after the start of casein injections are shown in Table 5. Of the 16 mice undergoing autopsy, only 3 (dead at 112 days) were found to have splenic amyloid; two were graded 0.25+ for amyloid, and the third l + . Unlike deaths in Experiment A which seemed to occur a t random among the four groups, those in Experiment B tended to occur earlier in the treated groups than in the controls. Beginning a t age 124 days, 2 control mice (one from each
205
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS
SANTWIN
CONTROL
Fig. 1. Plasma glycoproteinsseparated by cellulose acetate electrophoresis:effect of adding Santoquin to the diet.
control group) were killed at weekly intervals and splenic sections prepared (stained with H & E). To shorten the study, it was planned to perform autopsies on all the mice when a significant degree of splenic amyloidosis was observed in the control mice. The first 4 mice undergoing autopsy a t 124 and 131 days of age were free of splenic amyloidosis. However, a t age 138 days, both control splenic sections were graded 3+ for amyloid. At age 140 days, all the mice remaining in the four groups were killed. Plasma samples were not obtained as the possible significance of the a,-glycoprotein “X”peak seen in Experiment A was not appreciated at the time. Splenic sections were prepared and graded for amyloidosis as before. The data are presented in Table 6. Amyloid was significantly less (P < 0.05; T test, excluding mice without splenic amyloidosis) in the Santoquin and vitamin E groups as compared to the corresponding control groups. The mice in the Santoquin control group had more splenic amyloidosis than those in the vitamin E control group. This difference was largely due to a higher degree of amyloidosis in the mice in one cage of the Santoquin control group; numerous workers have pointed out that the susceptibility of mice to amyloidosis is affected by many undefined environmental factors (12).
effective as well in casein-induced amyloidosis, but the efficacies of vitamin E and BHT were reversed; vitamin E had a modest beneficial influence while BHT had little, if any. The differences in the actions of BHT and vitamin E on spontaneous and casein-induced amyloidosis may be related, a t least in part, to mouse-strain differences. Santoquin, and to a lesser extent BHT, was shown to depress the appearance of a plasma protein component(s) in the a,-globulin region in the casein-injected mice; the effect of a-tocopherol acetate was not determined. The action of antioxidants on amyloidosis implicates a free radical reaction(s) in the pathogenesis of this disorder. How such a reaction(s) may be involved is suggested by a consideration of current knowledge of amyloid in the light of antioxidant studies. Amyloid substance consists of a t least two fractions, both related to normal serum proteins. The plasma component (P-component) (13), a globular protein antigenically identical to an (Y ,-globulin present in normal human plasma (14), is a minor constituent of amyloid. The P-component has not been identified in tissue, only in partially purified amyloid preparations (13, 15). The amyloid fiber is the major component of amyloid (16-19). Two major types of proteins, now named (20) AL
DISCUSSION The average lifespan of male LAF, mice fed a semisynthetic diet containing 2 0 % casein ~ as the sole source of protein was increased 72 percent by adding 0.25% Santoquin to the diet, 18 percent by 0 . 2 5 % ~BHT and 3 percent by 0 . 5 0 % ~a-tocopherol acetate (7). The increase in the lifespan of the Santoquin and BHT groups is attributed in part to a depressing effect of the antioxidants on amyloid formation, this effect presumably being greater with Santoquin. In the present study Santoquin was found to be
Fig. 2. Plasma glycoproteins separated by polyacrylamide gel electrophoresis: effect of adding Santoquin to the diet.
207
VOl. XXIV
HARMAN, EDDY AND NOFFSINGER TABLE 4
Experiment B-Effect of Antioxidants on Casein-ZnducedAmyloidosis in Male C3HeBIFeJ Mice: Avemge Body Weight and Food Consumption
Age (days) 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140
Vitamin E 0 . 2 5 % ~
Control 1'
Santoquin 0 . 2 5 % ~
No.
Avg. Wt.
Food Intake"
No.
Avg. Wt.
Food Intake
40 40 40 40 40 40 40
17.6 21.1 22.8 22.5 23.6 24.2 24.1
14.0 23.2 25.2 19.8 19.2 19.8 18.5
50 50 50 50 50 50 50
17.0 21.6 23.2 23.5 24.0 24.1 24.2
15.7 25.1 23.3 19.5 17.3 17.9 17.7
29 29 29 29
27.3 28.3 28.9 29.9
23.4 26.2 24.2 24.0
45 43 42 41
28.2 29.1 29.4 30.2
24.4 25.1 23.6 24.0
Control 2"
No.
Avg. Wt.
Food Intake
No.
Avg. Wt.
Food Intake
40 40 40 40 40 40 39 39 37 35 33 33 33 32 32
19.2 21.7 23.2 23.1 24.1 24.7 25.1 25.8 27.6 28.3 28.6 29.5 29.8 30.5 31.2
18.5 21.3 23.2 19.8 19.2 19.4 19.5 19.6 24.2 21.4 26.6 24.9 23.9 23.6 24.1
50 50 50 50 50 50 50 47 46 45 45 42 41 39 38
18.8 21.7 23.0 23.3 24.1 24.3 24.5 25.5 27.1 28.0 28.1 29.4 29.8 30.6 30.7
18.8 23.4 21.8 19.8 18.3 17.4 17.5 18.0 22.1 21.8 23.6 24.4 23.6 23.9 24.1
Food/mouse/week. Casein injections started. 'Pair-fed control for the 0 . 2 5 % ~Santoquin group. vitamin E group. Pair-fed control for the 0 . 2 5 % ~ a
TABLE 5
Experiment B-Effect of Antioxidants on Casein-Znduced Amyloidosis in Male C3HeBIFeJ Mice: Data on Mice Dying after Casein Injections Were Started at 77 Days of Age ~
Santoquin (days)
No. Dead
77 84
6 9 6 9 7 8
91 98
Vitamin E
Control 1 Cage
No.Dead
Cage
I
4" 1 1 2 lb
2
105 112
1 2 1 1
Control 2
No. Dead
15
2
10 13 14 14
17 18 15
1
13
1
10 11 13 14 10 13 11
1 l(l+)d
Total number/ mice that died1 in each group
1
lo
I
1 lb
lb
2E
1 12
lb
lb
119 126 6
No. Dead
1
l6
1(0.25+)' 1(0.25+)?
133
Cage
lb lb
1 lb
1
~~
8
12
Two mice underwent autopsy. Autopsy. 'One autopsy. 'Spleen amyloid, graded on basis of 0-4+.
(amyloid L chain) and AA (amyloid A), have been isolated from the fibers; the relative amounts of AL and AA depend upon the amyloid source. The AL group of proteins (21), found mainly in patients with primary amyloidosis and amyloidosis
208
associated with multiple myeloma, consists of fragments of immunoglobulin light chains which range in molecular weight from 5,000 to 25,000 daltons. The AL proteins may be derived, at least in part, from a,-globulins by proteolysis, as
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS
TABLE 6 Experiment B-Effect of Antioxidants on Casein-ZnducedAmyloidosis in Male C3HeBIFeJ Mice: Spleen Amyloid Spleen Amyloid" 0.0 0.1 0.25 0.5 1.o 1.5 2.0 2.5 3.0 3.5 -
Total spleen sections Total number of autopsies Number of spleen sections not available for grading
Santoquin
Control Ib
Vitamin E
Control 2"
18 2 1 1 1
18 1
23
23 1 2
2 1 1
2 4 5 4 36 40 4
27 28 1
1 1
1 2 1 2
2 31 32 1
2 1 3 1 1 1 35 38 3
* Spleen amyloid, graded on basis of &4+. Pair-fed control for the Santoquin group. Pair-fed control for the vitamin E group.
amyloid-like fibrils have been prepared from Bence-Jones proteins in this manner (22). Protein AA (23-25) is not antigenically related to any known immunoglobulin. The AA protein generally has a molecular weight of 8500 daltons. Protein SAA (amyloid A-related serum component), apparently usually present in very small amounts in the al-globulin region of normal human serum, is antigenically related to protein AA (26, 27). The molecular weight of protein SAA is about 100,000 daltons. Protein SSA, i.e., the serum nonimmunoglobulin-related amyloid component, is found with increased frequency (26-29) both with advancing age and a variety of chronic disorders associated with amyloidosis (e.g., tuberculosis, rheumatoid arthritis, lymphoma, leukemia, cancer and myeloma) as well as in all types of amyloidosis. Protein SSA increases in acute infections and inflammatory processes, and decreases quickly as the diseases are controlled. Elevated levels of protein SSA are common in pregnant women. All of the foregoing diverse conditions of elevated protein SSA are associated with elevated levels of serum copper (30). About 90 percent of serum copper is present as ceruloplasmin, a true oxidase, and most of the remaining 10 percent is loosely bound to protein (mainly albumin). Copper is an excellent catalyst for the reaction of molecular oxygen with organic compounds (31). Increases in the level of serum copper are likely accompanied by increases in the rate of reaction of molecular oxygen with tissue constituents, both of cells and of connective tissue. Judging from studies of the oxidative degradation of hyaluronic
acid (32), the turnover of protein polysaccharides of connective tissue (33, 34) may be due in part to a reaction with molecular oxygen. The rate of this reaction might be expected to be enhanced by increasing the level of serum copper or by altering other rate-controlling factors. From this point of view, the a,-glycoprotein "X" peak observed in Experiment A may be the result of oxidative degradation of connective-tissue components, a process depressed by both Santoquin and BHT. Thus SSA may be derived from connectivetissue components which subsequently, through enzymaticloxidative change, are further degraded to protein AA which, in t u n , becomes aggregated to form amyloid fibrils. By analogy, the AL proteins and the P-component of amyloid may be formed by a similar process from the serum alimmunoglobulin precursors. The beneficial effect of antioxidants on amyloidosis may now be tentatively attributed to a decreased rate of oxidative degradation of tissue and serum constituents. The long induction period before amyloid is observed, at least in casein-induced murine amyloidosis, followed by the rapid, autocatalytic-like deposition of amyloid, may be related to a gradual depletion of inhibitors of tissue free-radical reactions. Aclznow ledgment The authors are indebted to Ann Eddy for her skilled and conscientious assistance in the casein-inducedamyloid experiments.
REFERENCES 1. Schwartz P.: Amyloidosis. Springfield, Illinois, Charles C Thomas, Publisher, 1970.
209
HARMAN, EDDY AND NOFFSINGER 2. Cohen AS: Amyloidosis, New England J Med 277: 522, 574, 628, 1967. 3. Glenner GG, Page D, Isersky C et al: Murine amyloid fibril protein: isolation, purification and characterization, J Histochem Cytochem 19: 16, 1971. 4. Harman D: Free radical theory of aging, Triangle 12: 153, 1973. 5. Heston WE and Deringer MK: Hereditary renal disease and amyloidosis in mice, Arch Path 46: 49, 1948. 6. Harman D: Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rat? of male LAF, mice, J Gerontol 23: 476, 1968. 7. Harman D: Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rate of male LAF, mice; second experiment, Gerontologist 8 (111): 13, 1968. 8. West WT: Experimental amyloidosis in mice: effect of high and low protein diets, J Nutrition 95: 323, 1968. 9. Pearce RH, Watson EM, Stodolski R et al: Effects of age and sex on the electrophoretic fractions of the serum glycoprotein, Clin Chem 10: 1066, 1964. 10. Clarke JT: Simplified disc (polyacrylamide gel) electrophoresis, Ann New York Acad Sci 121: 428, 1964. 11. Keyser JW: Staining of serum glycoproteins after electrophoretic separation in acrylamide gels, Analyt Biochem 9: 249, 1964. 12. Franklin EC and Clerici E: Amyloid, Progr Immunol 11. 4: 381, 1974. 13. Bladen HA, Nylen MU and Glenner GG: The ultrastructure of human amyloid as revealed by the negative staining technique, J Ultrastruct Res 14: 449, 1966. 14. Skinner M, Cohen AS, Shirahama T et al: P-component (pentagonal unit) of amyloid: isolation, characterization, and sequence analysis, J Lab Clin Med 84: 604, 1974. 15. Franklin EC and Zucker-Franklin D: Current concepts of amyloid, Adv Immunol 15: 249, 1972. 16. Shirahama T and Cohen AS: High-resolution electron microscopic analysis of the amyloid fibril, J Cell Biol 33: 679, 1967. 17. Pras M, Schubert M, Zucker-Franklin D et al: The characterization of soluble amyloid prepared in water, J Clin Invest 47: 924, 1968. 18. Glenner GG, Cuatrecasas P, Isersky C et al: Physical and chemical properties of amyloid fibers. II. Isolation of a unique protein constituting the major component from human splenic amyloid fibril concentrates, J Histochem Cytochem 17: 769, 1969.
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VOl. X X N
19. Harada M, Isersky C, Cuatrecasas P e t al: Human amyloid protein: chemical variability and homogeneity, J Histochem Cytochem 19: 1, 1971. 20. Wegelius 0 (Ed.): Amyloid Symposium (1976). New York, Academic Press Inc. (in press). 21. Glenner GC, Terry WD and Isersky C: Amyloidosis: its nature and pathogenesis, Seminars Hematol. 10: 65, 1973. 22. Linke RP, Zucker-Franklin D and Franklin EC: Morphologic, chemical, and immunologic studies of amyloidlike fibrils formed from Bence Jones proteins by proteolysis, J Immunol 111: 10, 1973. 23. Benditt EP and Eriksen N: Chemical classes of amyloid substance, Am J Path 65: 231, 1971. 24. Husby G, Sletten K. Michaelsen TE et al: Antigenic and chemical characterization of nonimmunoglobulin amyloid proteins, Scandinav J Immunol 1: 393, 1972. 25. Husby GK, Sletten K, Michaelsen T E e t al: Amyloid fibril protein subunit, protein AS, distribution in tissue and serum in different clinical types of amyloidosis including that associated with myelomatosis and Waldenstr6m’s macroglobulinaemia, Scandinav J Immunol 2: 395, 1973. 26. Levin M, Pras M and Franklin EC: Immunologic studies of the major nonimmunoglobulin protein of amyloid, J Exper Med 138: 373, 1973. 27. Husby GK, Natvig JB, Michaelsen T E et al: Unique amyloid protein subunit common to different types of amyloid fibril, Nature 244: 362, 1973. 28. Husby G and Natvig JB: A serum component related to nonimmunoglobulin amyloid protein AS, a possible precursor of the fibrils, J Clin Invest 53: 1054, 1974. 29. Fbsenthal CJ and Franklin EC: Variation with age and disease of an amyloid A protein-related serum component, J Clin Invest 55: 746, 1975. 30. Underwood EJ: Copper, in Trace Elements in Human and Animal Nutrition (3rd Ed.). New York, Academic Press, 1974, pp 57-115. 31. Ingold KW: Metal catalysis, in Symposium on Foods: Lipids and Their Oxidation, ed. by HW Schultz, EA Day and RO Sinnhuber. Westport, Conn., Avi Publishing Co, Inc, 1962, pp 93-121. 32. Pigman W, Rizvi S and Holley HL: Depolymerization of hyaluronic acid by the ORD reaction, Arth & Rheum 4: 240, 1961. 33. Schubert M and Harmerman D: A Primer on Connective Tissue Biochemistry. Philadelphia, Lea & Febiger, 1968. 34. Spiro RG: Glycoproteins, Adv Protein Chem 27: 349, 1973.
JOURNAL OF THE AMERICAN GERIATRICS SOCIETY Copyright 0 1976 by the American Geriatrics Society
Free Radical Theory of Aging: Inhibition of Amyloidosis in Mice by Antioxidants; Possible Mechanism* DENHAM HARMAN, MD, PhD**, DENNIS E. EDDY, PhD and JAMES NOFFSINGER, PhD Departments of Biochemistry and Medicine, University of Nebraska College of Medicine, Omaha, Nebraska
ABSTRACT: The antioxidants, a-tocopherol acetate and a quinolone derivative (Santoquin), inhibited the development of amyloidosis when added to the diet of casein-injected C3HeB/FeJ male mice. Santoquin, and to a lesser extent butylated hydroxytoluene (BHT), also depressed the appearance of a plasma protein fraction in these mice; the effect of a-tocopherol was not determined. Consideration of the current knowledge of amyloid, in the light of these antioxidant studies, prompted the following hypothesis. Amyloidosis is largely the result of an enhanced rate of oxidative degradation of a connectivetissue glycoprotein(s) coupled with oxidative/enzymatic changes in the plasma, both of the tissuederived substances and of immunoglobulins, to form the amyloid fibril protein subunits (ALand AA) which subsequently aggregate to form the amyloid fibrils.
The incidence of amyloidosis increases with age (1, 2). The reason for this association, as well as the pathogenesis of amyloidosis, is unknown. Amyloid is associated with a large number of seemingly unrelated diseases (2), both in man and in many species of animals. It can be induced in animals by a wide variety of methods (2, 3). Amyloid material from different sources has essentially the same appearance (2, 3). Under the light-microscope, it appears as an amorphous, hyaline, extracellular material, readily detected by its characteristic staining properties. In the electron-microscope, morphologically unique fibrils appear as the major constituent of amyloid. An observation made during the course of a study of the effect of antioxidants on the lifespan of male LAF, mice, based on the possibility that endogenous free radical reactions contribute to the degradation of biologic systems (4),may aid in shedding light on both the etiology of amyloidosis and its association with age. * The authors gratefully acknowledge the financial assistance of the late Mrs. Adah Millard of Omaha, Nebraska and of Mr. Myron Pfeifer of Concord, Tennessee. * * Address for correspondence: Denham Harman, MD, Department of Biochemistry, College of Medicine, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, NB 68105.
LAF, mice are prone to develop amyloidosis (5). Marked deposits of amyloid are found in the spleen, liver and kidneys of many mice of this strain a t death. The incidence of amyloidosis at death is diet-dependent. Mice fed a commercial pelleted diet have about a 25 percent incidence (5) of amyloidosis, but an approximately 65 percent incidence (6) if given a semisynthetic diet containing 2 0 % ~ (percent by weight) casein as the sole source of protein. The average lifespan of male LAF, mice fed a 2 0 % ~casein diet was markedly increased by adding to the diet 0.25-0.50%w of butylated ~ 1,2-dihyhydroxytoluene (BHT) (6) or 0 . 2 5 % of dro-6-ethoxy-2,2,4-trimethyquinoline(7) (Santoquin, Ethoxyquin-Monsanto Chemical Company, St. Louis, Missouri); another antioxidant, a-tocopherol acetate, had little if any effect (7). The increase in lifespan produced by the two antioxidants was paralleled by a decrease in the incidence of amyloidosis. This observation suggested that the beneficial effect of the antioxidants on average lifespan was due, at least in part, to inhibition of a free radical reaction(s) involved in amyloidosis. The purpose of the present study was to determine if casein-induced mouse amyloidosis would
203
VOl. XXIV
HARMAN, EDDY AND NOFFSINGER
distilled water for 48 hours; the water was changed frequently. The solution was then transferred to an ultra filtration apparatus (Amicon Corp., Lexington, Massachusetts), and concentrated to 25 ml by means of an Amicon Type XMlOOA membrane. The final 10% casein solution was stored in the cold room until use; storage time did not exceed 10 days.
also be inhibited by the addition of an antioxidant to the diet. Santoquin, BHT and a-tocopherol acetate were evaluated. METHODS AND RESULTS Two casein-induced amyloid experiments were carried out in sequence on C3HeBPeJ male mice. The mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) shortly after weaning, and then caged 10 per cage (stainless steel, 12x8 x 5 inches). They were maintained in an airconditioned room a t 76-78' F a t a humidity of 50-60 percent. The cages were changed twice a week. The bedding consisted of sterilized shredded corn cobs (San-i-Cel, Paxton Processing Company, Paxton, Illinois). Light was from overhead fluorescent lamps automatically controlled to provide light from 6:OO a.m. to 8:OO p.m. The mice were fed a commercial pelleted mouse chow (Rockland; Teklad, Inc., Monmouth, 11linois) from the time of arrival until they were about 40 days of age, when the experiments were started. Amyloidosis was induced by the subcutaneous injection of 0.3 ml of a 1 0 % (percent ~ by weight) aqueous solution of casein (8) 5 days per week, starting around 75 days of age. The casein solution was prepared by dissolving 2.5 gm of vitaminfree test casein (Teklad Mills, Madison, Wisconsin) in 50 ml of 0.25% NaOH. The casein solution was next dialyzed a t 4°C (cold room) against
Experiment A-Effect
50
57 63 71 7Sb
86 92 99 1 W 113 120 126 134 141 148 154
No.
Avg. Wt.
30 30 30 30 30 30 30 29 29 28 24 20 16 12 7 5
18.4 21.3 22.4 23.4 24.2 24.7 24.7 26.0 26.6 27.4 27.9 27.8 28.6 27.7 28.1 27.8
0
In the first experiment, 120 mice were divided into four dietary groups of 30 each, a t age 43 days. They were fed a powdered diet (Rockland) to which was added 0 . 2 5 % ~of BHT, or 0 . 2 5 % ~of Santoquin, or nothing (the pair-fed controls for the antioxidant groups). A t 78 days of age, subcutaneous injection of 1 0 % casein ~ solution was started (0.3 ml per mouse five days per week). Food consumption and body weight data are presented in Table 1. There were no significant differences between the four groups except for a modest decrease in food consumption in the BHT group beginning a t age 134 days. Data on the 7 mice that died after the start of casein injections are shown in Table 2. Starting a t 106 days of age, a t short intervals 2 mice from each group underwent autopsy until the end of the experiment a t age 155 days. This autopsy schedule was prompted by an initial study employing 50 mice; splenic sections obtained before 120 days of age were free of amyloid whereas after
TABLE 1 of Antioxidants on Casein-Induced Amyloidosk in Male C3HeBIFd Mice: Average Body Weight and Food Consumption Control Id
BHT 0.25%~
43
Experiment A
-
Control T
Santoquin 0.25%~
Food Intake'
No.
Avg. Wt.
Food Intake
13.7 20.5 19.5 19.5 17.6 17.4 17.9 20.3 20.3 21.0 19.6 20.3 18.9 17.5 18.2 14.0
30 30 30 30 30 30 29 29 29 29 25 21 17 13 9 7 3
19.0 22.7 23.7 24.5 25.0 25.4 26.2 26.8 27.4 28.0 28.2 28.5 29.1 29.5 30.1 30.0 27.7
18.4 20.8 19.4 20.3 19.0 18.7 19.0 20.2 21.2 22.4 21.7 22.3 24.5 24.5 23.1 23.1
No.
30 30 30 30 30 30 30 30 29
29 25 21 17 13 9 7 3
Avg. Wt.
18.4 22.1 23.0 23.8 24.6 24.9 25.5 26.0 26.4 27.2 27.4 27.2 27.8 28.1 27.8 26.7 27.7
Food Intake
17.4 21.2 20.5 20.9 17.4 17.9 19.6
21.6 19.6 21.0 21.0 22.3 21.7 21.0 19.5 21.0
Avg.
No.
Wt.
30 30 30 30 30 30 30 29 28
18.0 21.6 22.9 24.0 24.4 24.9 25.7 26.2 26.9 27.1 27.8 28.1 28.3 27.8 27.0 27.0 25.0
28 24 20 16 12 8 6 2
Control 3'
Food Intake
18.7 20.6 18.6 18.5 17.0 17.9 18.0 19.9
20.3 22.2 20.3 21.7 22.4 18.9 18.6 16.8
No. 10 10 10 10 10 10 10 10 10 10 8
7 6 5 4 4 4
Avg. Wt.
17.9 21.6 23.1 24.2 24.2 24.6 25.8 26.0 26.6 27.3 27.9 28.1 28.1 28.2 28.2 28.2 28.5
Food Intake
18.2 22.5 19.9 18.2 17.2 18.4 18.6 20.6 19.1 21.3 18.9
21.0 23.8 25.2 22.4 28.0
Foodhnouselweek. Casein iniections started. At age 106 days.4 animals were killed per week in the first 4 groups and 1 animal per week in Control 3 (water-injected grcup), slides made, and degree of amyloidosis noted. Pair-fed control for the 0.25%~BHT group. 'Pair-fed control for the 0.25%~Santoquin group. ' Pair-fed with the Santoquin group, injected with distilled water, a
204
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS TABLE 2 Experiment A-Effect of Antioxidants on Casein-Induced Amyloidosis in Male C3HeBIFeJ Mice: Data on Mice Dying after Casein Injections Were Started at 78 Days of Age Santoquin
Age (days)
Cage
Control 1
No. Dead
85 92 98
99
8
BHT
Cage
No. Dead
4
1‘
Cage
No. Dead,
Cage
No. Dead
2
1
11 11
1 1
3 1
1 (3+P 1 (2+P
1.
134 149 a
Control 2
No autopsy. Spleen amyloid, graded on basis of 0-4+
age 150 days, when autopsies were resumed, all splenic sections were about the same (3.5+ for amyloid). Sections of spleen and liver were stained with hemotoxylin and eosin (H & E). The spleen sections were graded on three separate occasions for amyloid (on the basis of 0-4+) by the same individual and the average of the three gradings recorded; the observer did not know the identity of the slides. Initially the liver sections were also graded for amyloidosis; the gradings were stopped as they were parallel to, but less than, the splenic values. The splenic gradings are shown in Table 3. The incidence of splenic amyloidosis in the BHT-treated group was not significantly different from that in its pair-fed control group, although some BHT mice in the latter part of the experiment were free of amyloid whereas none of the controls were after age 134 days. In contrast, the Santoquin group had significantly less (P < 0.05; Mann-Whitney U test) splenic amyloidosis than the control group. A blood sample was obtained from each mouse at autopsy and the plasma (EDTA) subjected to cellulose acetate electrophoresis (9); the strips were stained for glycoprotein (9) and the relative amounts of each fraction measured by densitometry. The densitometer tracings were all similar except for a component (a-glycoprotein “X”) in the a,-region indicated by an arrow in Figure 1. This component, seen first at 109 days, was observed with increasing frequency as the number of casein injections increased, but tended to be less in the Santoquin and BHT groups. The a,-glycoprotein “X” peak was also observed in the plasma of water-injected mice (pair-fed with the Santoquin group), beginning at age 130 days. The proportion of mice in each group with the plasma glycoprotein “X” peak was: BHT, 34 percent (10/29); BHT control, 83 percent (24/29); Santoquin, 17 percent (5/29); Santoquin control,
64 percent (16/25); and sterile-water injected controls, 60 percent (6/10). In Table 3 the area of the small peak in the a,-glycoprotein region (see Figure 1) is listed in the section below the corresponding splenic gradings. A few plasma (EDTA) samples were available for polyacrylamide gel electrophoresis (10); the gels were stained for glycoprotein (11). A band in the a,-globulin region was decreased (indicated by the arrow in Figure 2) in the plasma of several mice that had received Santoquin in their diet; this decrease was not observed in the one sample of plasma run from the BHT group.
Experiment B Experiment B was carried out to confirm the Santoquin results of Experiment A and to evaluate the effect of a-tocopherol acetate on caseininduced amyloidosis. For the second study, 180 mice were divided into four groups, 40 in each of two treated groups and 50 in each of two control groups, at age 35 days, and given a powdered diet to which was added 0 . 2 5 % Santoquin, ~ or 0 . 2 5 % ~ a-tocopherol acetate, or nothing (the pair-fed controls for the two antioxidant groups). At 77 days of age, subcutaneous injection of casein solution was begun, as described in Experiment A. Weekly food consumption and body weight data are shown in Table 4. No significant differences were noted. Data on the 42 mice that died after the start of casein injections are shown in Table 5. Of the 16 mice undergoing autopsy, only 3 (dead at 112 days) were found to have splenic amyloid; two were graded 0.25+ for amyloid, and the third l + . Unlike deaths in Experiment A which seemed to occur a t random among the four groups, those in Experiment B tended to occur earlier in the treated groups than in the controls. Beginning a t age 124 days, 2 control mice (one from each
205
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS
SANTWIN
CONTROL
Fig. 1. Plasma glycoproteinsseparated by cellulose acetate electrophoresis:effect of adding Santoquin to the diet.
control group) were killed at weekly intervals and splenic sections prepared (stained with H & E). To shorten the study, it was planned to perform autopsies on all the mice when a significant degree of splenic amyloidosis was observed in the control mice. The first 4 mice undergoing autopsy a t 124 and 131 days of age were free of splenic amyloidosis. However, a t age 138 days, both control splenic sections were graded 3+ for amyloid. At age 140 days, all the mice remaining in the four groups were killed. Plasma samples were not obtained as the possible significance of the a,-glycoprotein “X”peak seen in Experiment A was not appreciated at the time. Splenic sections were prepared and graded for amyloidosis as before. The data are presented in Table 6. Amyloid was significantly less (P < 0.05; T test, excluding mice without splenic amyloidosis) in the Santoquin and vitamin E groups as compared to the corresponding control groups. The mice in the Santoquin control group had more splenic amyloidosis than those in the vitamin E control group. This difference was largely due to a higher degree of amyloidosis in the mice in one cage of the Santoquin control group; numerous workers have pointed out that the susceptibility of mice to amyloidosis is affected by many undefined environmental factors (12).
effective as well in casein-induced amyloidosis, but the efficacies of vitamin E and BHT were reversed; vitamin E had a modest beneficial influence while BHT had little, if any. The differences in the actions of BHT and vitamin E on spontaneous and casein-induced amyloidosis may be related, a t least in part, to mouse-strain differences. Santoquin, and to a lesser extent BHT, was shown to depress the appearance of a plasma protein component(s) in the a,-globulin region in the casein-injected mice; the effect of a-tocopherol acetate was not determined. The action of antioxidants on amyloidosis implicates a free radical reaction(s) in the pathogenesis of this disorder. How such a reaction(s) may be involved is suggested by a consideration of current knowledge of amyloid in the light of antioxidant studies. Amyloid substance consists of a t least two fractions, both related to normal serum proteins. The plasma component (P-component) (13), a globular protein antigenically identical to an (Y ,-globulin present in normal human plasma (14), is a minor constituent of amyloid. The P-component has not been identified in tissue, only in partially purified amyloid preparations (13, 15). The amyloid fiber is the major component of amyloid (16-19). Two major types of proteins, now named (20) AL
DISCUSSION The average lifespan of male LAF, mice fed a semisynthetic diet containing 2 0 % casein ~ as the sole source of protein was increased 72 percent by adding 0.25% Santoquin to the diet, 18 percent by 0 . 2 5 % ~BHT and 3 percent by 0 . 5 0 % ~a-tocopherol acetate (7). The increase in the lifespan of the Santoquin and BHT groups is attributed in part to a depressing effect of the antioxidants on amyloid formation, this effect presumably being greater with Santoquin. In the present study Santoquin was found to be
Fig. 2. Plasma glycoproteins separated by polyacrylamide gel electrophoresis: effect of adding Santoquin to the diet.
207
VOl. XXIV
HARMAN, EDDY AND NOFFSINGER TABLE 4
Experiment B-Effect of Antioxidants on Casein-ZnducedAmyloidosis in Male C3HeBIFeJ Mice: Avemge Body Weight and Food Consumption
Age (days) 35 42 49 56 63 70 77 84 91 98 105 112 119 126 133 140
Vitamin E 0 . 2 5 % ~
Control 1'
Santoquin 0 . 2 5 % ~
No.
Avg. Wt.
Food Intake"
No.
Avg. Wt.
Food Intake
40 40 40 40 40 40 40
17.6 21.1 22.8 22.5 23.6 24.2 24.1
14.0 23.2 25.2 19.8 19.2 19.8 18.5
50 50 50 50 50 50 50
17.0 21.6 23.2 23.5 24.0 24.1 24.2
15.7 25.1 23.3 19.5 17.3 17.9 17.7
29 29 29 29
27.3 28.3 28.9 29.9
23.4 26.2 24.2 24.0
45 43 42 41
28.2 29.1 29.4 30.2
24.4 25.1 23.6 24.0
Control 2"
No.
Avg. Wt.
Food Intake
No.
Avg. Wt.
Food Intake
40 40 40 40 40 40 39 39 37 35 33 33 33 32 32
19.2 21.7 23.2 23.1 24.1 24.7 25.1 25.8 27.6 28.3 28.6 29.5 29.8 30.5 31.2
18.5 21.3 23.2 19.8 19.2 19.4 19.5 19.6 24.2 21.4 26.6 24.9 23.9 23.6 24.1
50 50 50 50 50 50 50 47 46 45 45 42 41 39 38
18.8 21.7 23.0 23.3 24.1 24.3 24.5 25.5 27.1 28.0 28.1 29.4 29.8 30.6 30.7
18.8 23.4 21.8 19.8 18.3 17.4 17.5 18.0 22.1 21.8 23.6 24.4 23.6 23.9 24.1
Food/mouse/week. Casein injections started. 'Pair-fed control for the 0 . 2 5 % ~Santoquin group. vitamin E group. Pair-fed control for the 0 . 2 5 % ~ a
TABLE 5
Experiment B-Effect of Antioxidants on Casein-Znduced Amyloidosis in Male C3HeBIFeJ Mice: Data on Mice Dying after Casein Injections Were Started at 77 Days of Age ~
Santoquin (days)
No. Dead
77 84
6 9 6 9 7 8
91 98
Vitamin E
Control 1 Cage
No.Dead
Cage
I
4" 1 1 2 lb
2
105 112
1 2 1 1
Control 2
No. Dead
15
2
10 13 14 14
17 18 15
1
13
1
10 11 13 14 10 13 11
1 l(l+)d
Total number/ mice that died1 in each group
1
lo
I
1 lb
lb
2E
1 12
lb
lb
119 126 6
No. Dead
1
l6
1(0.25+)' 1(0.25+)?
133
Cage
lb lb
1 lb
1
~~
8
12
Two mice underwent autopsy. Autopsy. 'One autopsy. 'Spleen amyloid, graded on basis of 0-4+.
(amyloid L chain) and AA (amyloid A), have been isolated from the fibers; the relative amounts of AL and AA depend upon the amyloid source. The AL group of proteins (21), found mainly in patients with primary amyloidosis and amyloidosis
208
associated with multiple myeloma, consists of fragments of immunoglobulin light chains which range in molecular weight from 5,000 to 25,000 daltons. The AL proteins may be derived, at least in part, from a,-globulins by proteolysis, as
May 1976
INHIBITION OF AMYLOIDOSIS BY ANTIOXIDANTS
TABLE 6 Experiment B-Effect of Antioxidants on Casein-ZnducedAmyloidosis in Male C3HeBIFeJ Mice: Spleen Amyloid Spleen Amyloid" 0.0 0.1 0.25 0.5 1.o 1.5 2.0 2.5 3.0 3.5 -
Total spleen sections Total number of autopsies Number of spleen sections not available for grading
Santoquin
Control Ib
Vitamin E
Control 2"
18 2 1 1 1
18 1
23
23 1 2
2 1 1
2 4 5 4 36 40 4
27 28 1
1 1
1 2 1 2
2 31 32 1
2 1 3 1 1 1 35 38 3
* Spleen amyloid, graded on basis of &4+. Pair-fed control for the Santoquin group. Pair-fed control for the vitamin E group.
amyloid-like fibrils have been prepared from Bence-Jones proteins in this manner (22). Protein AA (23-25) is not antigenically related to any known immunoglobulin. The AA protein generally has a molecular weight of 8500 daltons. Protein SAA (amyloid A-related serum component), apparently usually present in very small amounts in the al-globulin region of normal human serum, is antigenically related to protein AA (26, 27). The molecular weight of protein SAA is about 100,000 daltons. Protein SSA, i.e., the serum nonimmunoglobulin-related amyloid component, is found with increased frequency (26-29) both with advancing age and a variety of chronic disorders associated with amyloidosis (e.g., tuberculosis, rheumatoid arthritis, lymphoma, leukemia, cancer and myeloma) as well as in all types of amyloidosis. Protein SSA increases in acute infections and inflammatory processes, and decreases quickly as the diseases are controlled. Elevated levels of protein SSA are common in pregnant women. All of the foregoing diverse conditions of elevated protein SSA are associated with elevated levels of serum copper (30). About 90 percent of serum copper is present as ceruloplasmin, a true oxidase, and most of the remaining 10 percent is loosely bound to protein (mainly albumin). Copper is an excellent catalyst for the reaction of molecular oxygen with organic compounds (31). Increases in the level of serum copper are likely accompanied by increases in the rate of reaction of molecular oxygen with tissue constituents, both of cells and of connective tissue. Judging from studies of the oxidative degradation of hyaluronic
acid (32), the turnover of protein polysaccharides of connective tissue (33, 34) may be due in part to a reaction with molecular oxygen. The rate of this reaction might be expected to be enhanced by increasing the level of serum copper or by altering other rate-controlling factors. From this point of view, the a,-glycoprotein "X" peak observed in Experiment A may be the result of oxidative degradation of connective-tissue components, a process depressed by both Santoquin and BHT. Thus SSA may be derived from connectivetissue components which subsequently, through enzymaticloxidative change, are further degraded to protein AA which, in t u n , becomes aggregated to form amyloid fibrils. By analogy, the AL proteins and the P-component of amyloid may be formed by a similar process from the serum alimmunoglobulin precursors. The beneficial effect of antioxidants on amyloidosis may now be tentatively attributed to a decreased rate of oxidative degradation of tissue and serum constituents. The long induction period before amyloid is observed, at least in casein-induced murine amyloidosis, followed by the rapid, autocatalytic-like deposition of amyloid, may be related to a gradual depletion of inhibitors of tissue free-radical reactions. Aclznow ledgment The authors are indebted to Ann Eddy for her skilled and conscientious assistance in the casein-inducedamyloid experiments.
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209
HARMAN, EDDY AND NOFFSINGER 2. Cohen AS: Amyloidosis, New England J Med 277: 522, 574, 628, 1967. 3. Glenner GG, Page D, Isersky C et al: Murine amyloid fibril protein: isolation, purification and characterization, J Histochem Cytochem 19: 16, 1971. 4. Harman D: Free radical theory of aging, Triangle 12: 153, 1973. 5. Heston WE and Deringer MK: Hereditary renal disease and amyloidosis in mice, Arch Path 46: 49, 1948. 6. Harman D: Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rat? of male LAF, mice, J Gerontol 23: 476, 1968. 7. Harman D: Free radical theory of aging: effect of free radical reaction inhibitors on the mortality rate of male LAF, mice; second experiment, Gerontologist 8 (111): 13, 1968. 8. West WT: Experimental amyloidosis in mice: effect of high and low protein diets, J Nutrition 95: 323, 1968. 9. Pearce RH, Watson EM, Stodolski R et al: Effects of age and sex on the electrophoretic fractions of the serum glycoprotein, Clin Chem 10: 1066, 1964. 10. Clarke JT: Simplified disc (polyacrylamide gel) electrophoresis, Ann New York Acad Sci 121: 428, 1964. 11. Keyser JW: Staining of serum glycoproteins after electrophoretic separation in acrylamide gels, Analyt Biochem 9: 249, 1964. 12. Franklin EC and Clerici E: Amyloid, Progr Immunol 11. 4: 381, 1974. 13. Bladen HA, Nylen MU and Glenner GG: The ultrastructure of human amyloid as revealed by the negative staining technique, J Ultrastruct Res 14: 449, 1966. 14. Skinner M, Cohen AS, Shirahama T et al: P-component (pentagonal unit) of amyloid: isolation, characterization, and sequence analysis, J Lab Clin Med 84: 604, 1974. 15. Franklin EC and Zucker-Franklin D: Current concepts of amyloid, Adv Immunol 15: 249, 1972. 16. Shirahama T and Cohen AS: High-resolution electron microscopic analysis of the amyloid fibril, J Cell Biol 33: 679, 1967. 17. Pras M, Schubert M, Zucker-Franklin D et al: The characterization of soluble amyloid prepared in water, J Clin Invest 47: 924, 1968. 18. Glenner GG, Cuatrecasas P, Isersky C et al: Physical and chemical properties of amyloid fibers. II. Isolation of a unique protein constituting the major component from human splenic amyloid fibril concentrates, J Histochem Cytochem 17: 769, 1969.
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