CRYOBIOLOGY
29, 668-673 (1992)
Deterioration of Cold-Stored Tissue Specimens Due to Lipid Peroxidation: Modulation by Antioxidants at High Subzero Temperatures G . S. W. WHITELEY, University Department
B. J. FULLER, AND K. E. F. HOBBS
of Surgery, Royal Free Hospital and School of Medicine, Hampstead, London NW3 2QG, United Kingdom
Pond Street,
It is often necessary to store tissue specimens in subzero conditions for assay in batches. During storage at -20°C we found that sufftcient lipid peroxidation occurred in rat liver homogenates in phosphate-buffered saline to affect subsequent malondialdehyde assays. This peroxidation did not occur at - 196°C. The ratio of oxidized to reduced glutathione increased with storage at - 20°C and the level of conjugated dienes increased progressively. The addition of a specific free radical scavenger, superoxide dismutase (200 u/ml) reduced the level of malondialdehyde (P < 0.001) during -20°C storage for periods of 28 days but failed to prevent the changes in the glutathione ratio or dienes. Storage in a less specific free radical scavenger, 0.25 molar sucrose/EDTA, instead of phosphatebuffered saline totally prevented the malondialdehyde production over similar storage periods. o 1992 Academic Press, Inc.
Lipid peroxidation due to the action of oxygen-derived free radical species on polyunsaturated fatty acids has been reported in liver tissue during unfavorable conditions. Insults including ischemia and reperfusion injury (1, 3) and the administration of toxins (4, 11) will lead to lipid peroxidation. Freezing is an insult which combines cellular disruption with chemical change. In many biochemical or pharmacological studies, liver samples or homogenates are frozen for subsequent assay. Since mammalian liver cell membranes are rich in polyunsaturated acids and the products of lipid peroxidation have many pharmacological actions by themselves, it is of interest to consider the possibility that freeze-storage of liver samples may lead to lipid peroxidation. Malondialdehyde is an end product of lipid peroxidation (5, 8) which can be measured by several techniques (2, 11, 13, 14). Conjugated dienes, produced at an early stage in the lipid peroxidation pathway to Received August 14, 1991; accepted July 3, 1992
malondialdehyde, can also be measured. The oxidation of reduced glutathione to oxidized glutathione is an important endogenous defense mechanism against free radical activity. The measurement of both oxidized and reduced glutathione acts as a third measure of free radical activity. This study shows that measurable lipid peroxidation takes place in frozen tissues which leads to tissue deterioration. More interestingly, the addition of a specific scavenging enzyme, superoxide dismutase, during the freezing period altered some aspects of this oxidative damage, while a more general scavenger, sucrose/EDTA, containing a sugar together with a chelating agent had a greater effect. MATERIALS
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS
The experiments used three groups of 10 male Sprague-Dawley rats weighing between 200 and 250 g. The rats were anesthetized using ether inhalation and hepatectomy was performed. Homogenization was performed as rapidly as possible in iced Dulbecco’s A phosphate-buffered saline
668 OOll-2240/92 $5.00
AND
COLD-STORED
TISSUE
DETERIORATION
(PBS) using a rotary PTFE plunger and glass tube. Homogenate (5 ml) samples were set aside as the control (control group A) for each test and an immediate assay for malondialdehyde (MDA) using a thiobarbituric acid reaction (14) was carried out on 10 specimens to act as a control. Simultaneous assays were performed for oxidized and reduced glutathione together with an assay for conjugated dienes. Homogenate was quick-frozen by rapid transition through nitrogen vapor into liquid nitrogen (liquid nitrogen storage group C) or frozen in a freezing cabinet at - 20°C ( - 20°C storage group B). Storage periods of 1, 7, and 28 days were studied. The assays were performed after the specified storage periods on 10 specimens in each experimental group. Samples of liver homogenate to which 200 u/ml of superoxide dismutase (from bovine erythrocytes) was added were stored concurrently at both temperatures (liquid nitrogen storage + SOD group D; -20°C storage + SOD group E). These samples were compared with the untreated homogenate. A further group of samples which had been homogenized in 0.25 molar sucrose/ EDTA instead of Dulbecco’s phosphatebuffered saline was concurrently stored at both temperatures (liquid nitrogen storage + sucrose/EDTA group F; -20°C storage + sucrose/EDTA group G). They were assayed after the same storage periods. A summary of the experimental groups follows: Group A, Control, freshly prepared homogenate in PBS. Group B, PBS homogenate frozen to -20°C and then stored. Group C, PBS homogenate frozen to - 196°C and then stored. Group D, - 196°C storage + SOD. Group E, -20°C storage + SOD. Group F, -’ 196°C storage + sucrose/ EDTA. Group G, -20°C storage + sucrose/ EDTA.
DUE TO LIPID
PEROXIDATION
669
Assay Methods
Prior to assay, all specimens were quickthawed in a 37°C water bath. The homogenates were mixed thoroughly on a rotary shaker and samples were immediately transferred to the assay reaction vials. Malondialdehyde was assayed using a thiobarbituric acid reaction (14) in duplicate lo-ml plain tubes with screw caps. Each tube contained 0.2 ml 7% sodium lauryl sulfate, 2.0 ml 0.1 molar hydrochloric acid, 0.3 ml 10% phosphotungstic acid, and 1.0 ml 0.67% thiobarbituric acid. Two milliliters of homogenate sample was added to each tube. A 16-nmol malondialdehyde standard was set up together with a reagent blank using 2 ml of Dulbecco’s A phosphatebuffered saline. The contents of the tubes were mixed and the tubes were incubated at 95°C for PBS samples and 85°C for sucrose samples (to prevent caramelization of the sucrose) in a water bath for 1 h. At the end of the incubation the tubes were placed on ice for 10 min. Ice-cold butan-l-01 (5 ml) was added. The tubes were well mixed and centrifuged at 3000 rpm for 10 min. The upper butan-l-01 layer was removed and read on a Perkin-Elmer LS5 fluorimeter using a 515nm excitation and a scanning emission between 540 and 560 nm. Conjugated dienes were estimated using extraction by 8 ml of a 2:l chloroform: methanol mixture to which 400 ~1 of liver homogenate had been added. Thorough mixing was performed followed by centrifugation at 2500 r-pm for 5 min. A blank using 400 ~1 of Dulbecco’s phosphate-buffered saline was set up. The samples were duplicated and read against the blank on an ultraviolet spectrophotometer at a wavelength of 240 nm. The reduced glutathione assay (6) was performed as follows. A 5O-kl sample of liver homogenate was diluted with 450 t.~lof phosphate EDTA buffer at pH 8. A IOO-~1 sample of this initial dilution was added to
670
WHITELEY,
FULLER,
3.7 ml of phosphate EDTA buffer in a lo-ml plain glass blood tube. To this mixture 200 ~1 of orthophthalaldehyde (0.02 g in 20 ml methanol) was and the contents were mixed. The tubes were incubated at room temperature for 15 min. All samples were assayed in duplicate against a standard and phosphate EDTA blank on a Perkin-Elmer LS5 fluorimeter at an excitation of 350 nm and a scanning emission between 410 and 440 nm. The oxidized glutathione (6) was assayed as follows. A 500-~1 sample of liver homogenate was mixed with 200 ~1 of 0.04 molar n-ethyl maleimide which had been freshly prepared. This mixture was incubated at room temperature for 30 min. To quench the reaction and bring the pH to 12, 4.3 ml of 0.1 M sodium hydroxide was added at the end of this period. An aliquot of 100 ~1 was taken and diluted with 3.7 ml of 0.1 M sodium hydroxide. Two hundred microliters of orthophthalaldehyde (0.02 g in 20 ml methanol) was added and the mixture incubated at room temperature for 15 min. The samples were read in duplicate against a standard and reagent blank at an excitation of 350 nm and a scanning emission between 410 and 440 nm. The malondialdehyde concentration was calculated in nmol/g liver. The glutathione ratio (oxidized/reduced) was calculated and the level of conjugated dienes were measured in fluorescence units per gram liver. Statistical analysis between groups was
AND HOBBS
performed using an unpaired Student t test. A statistical level of P < 0.01 or better was accepted for significance. RESULTS
The data for liver homogenate stored at - 20°C (group B) and - 196°C (group C) are shown in Table 1. The effect of storage temperature on malondialdehyde is shown in Fig. 1. The effects of superoxide dismutase (groups D and E) and sucrose/EDTA (groups F and G) on malondialdehyde production are shown in Fig. 1. A malondialdehyde assay was performed after 2 h freezing at both - 20 and - 196°C on 10 PBS homogenate samples to assess the effect of the freeze-thaw cycle. There was no significant difference in malondialdehyde production over control. Table 2 shows values for all measurements of peroxidation for all experimental groups at 1 and 28 days of storage. Malondialdehyde production was shown to occur in the liver tissue stored at - 20°C (group B) and was greater than the control (group A) level after only 24 h storage (P < 0.001). The level of malondialdehyde rose after 7 and 28 days storage to a significant level (P < 0.001). The level of malondialdehyde reached in the -20°C (group B) storage group was very high, reaching approximately five times the control level at 28 days. However, in the group stored at - 196°C (group C) the production of malondialdehyde in liver homogenates was suppressed.
TABLE 1 Values for MDA Content, Oxidized/Reduced Glutathione Ratio, and Conjugated Dienes in Liver Homogenates in PBS at -20°C (Group B) and - 196°C (Group C) during 28 Days Storage (means 2 SD)
Group B Day Day Day Day
0 1 7 28
81.1 178.2 337.4 445.8
+ 2 k 2
17.3 31.9* 31.9* 38.6*
* P < 0.001 from control.
Group C 81.1 68.0 66.0 93.3
k 2 2 2
Conjugated dienes fluorescence units/g
Oxidized/reduced glutathione ratio
MDA content (nmoYg)
17.3 16.6 13.3 12.0
Group B 0.41 1.09 1.69 1.43
2 ” 2 5
0.04 0.38* 0.22* 0.07*
Group C 0.41 0.94 0.99 1.08
f 2 2 ”
0.04 0.03* 0.10* 0.17*
Group B 6.8 7.7 8.7 10.4
* 2 + 2
0.4 0.8 0.9 0.6
Group C 6.8 8.6 8.5 8.1
f k * t
0.4 0.2 0.6 0.3
COLD-STORED
0
TISSUE
DETERIORATION
7
StoragePerti khys) .
FIG. 1. The effect of storage temperature on malondialdehyde production (nmol/g) in stored liver homogenate (mean + SD, n = 10).
Superoxide dismutase added prior to freezing reduced the level of malondialdehyde over control (P < 0.001) at 1,7, and 28 days storage at -20°C (group D). Nevertheless, superoxide dismutase did not totally prevent the rise in malondialdehyde level (Fig. 2). Malondialdehyde production was reduced by storage in 0.25 M sucrose/ EDTA (group F). There was no significant rise in malondialdehyde production at 1, 7, and 28 days storage at - 20°C over the control group.
DUE TO LIPID
PEROXIDATION
671
The glutathione ratio (oxidized/reduced) increased with the period of storage. The ratio reached a significant (P < 0.001) level at -20°C (group B) after 1 day. The values at -20°C were higher than those stored at - 196°C (groups C vs B) (P < 0.001) at all storage intervals. However, superoxide dismutase did not prevent the rise in glutathione ratio with increasing storage periods at - 20°C. In the group stored at - 20°C in sucrose/EDTA the glutathione ratio was no different than the group stored without antioxidant (groups F vs B). Conjugated diene level rose with the duration of storage at -20°C (group B) achieving a significantly high level at 28 days (P < 0.001). At - 196°C (group C) there was no significant increase in the level of conjugated dienes throughout the period of storage. Superoxide dismutase failed to prevent the rise in diene level in group B at -20°C. Sucrose/EDTA storage at -20°C (group F) reduced the level of conjugated dienes at 28 days (P < 0.001). The combined changes in MDA content, oxidized/reduced glutathione ratio, and conjugated diene levels in the experimental groups on Day 1 and Day 28 are shown in Table 2.
TABLE
DISCUSSION
From the above experiment we have 2
Values for MDA Content, Oxidized/Reduced Glutathione Ratio, and Conjugated Dienes (means f SD) in Liver Homogenates after 1 and 28 Days Storage MDA content (nmol/g)
Group B ( - 20°C storage) Group C (- 196°C storage) Group D (- 196°C + SOD) Group E (-20°C + SOD) Group F f - 196°C f SucroselEDTA) Group G ( - 20°C + Sucrose/EDTA) * P < 0.001 from control.
Oxidized/reduced glutathione ratio
Conjugated dienes (fluorescence/g)
Day 1
Day 28
Day 1
Day 28
Day 1
Day 28
178.2 + 31.9*
445.8 2 38.6*
1.09 + 0.38*
1.43 + 0.07’
7.7 + 0.8
10.4 + 0.6
68.0 k 16.6
93.3 2 12.0
0.94 i 0.03*
1.08 + 0.17*
8.6 ? 0.2
8.1 + 0.3
70.11 + 4.15
85.03 2 3.90
0.91 2 0.10*
1.51 2 0.07*
6.9 2 0.1
7.5 It 0.5
117.82 i 5.89*
370.46 ? 1I .05*
1.20 k 0.04*
2.01 5 0.12*
6.4 2 0.6
8.4 k 1.6
65.50 t 6.97
104.02 5 7.28
1.09 -c 0.05*
1.75 -c 0.05*
6.4 f 0.3
6.5 k 0.4
66.97 + 3.23
113.44 f 6.68
1.07 f 0.03;
1.60 k 0.06*
5.9 t 0.6
5.6 + 0.5
672
WHITELEY,
FULLER,
PBSalone(GroupB ) PBSt 200~I ml SODI GroupE 1 PBSt 0.25MSucroselEDTAl GroupG I
StoragePeriod(days)
FIG. 2. The effect of superoxide dismutase and sucrose/EDTA on malondialdehyde production (nmoVg) in liver homogenate stored at - 20°C (mean 2 SD, n = 10).
demonstrated that lipid peroxidation of liver homogenates as measured by malondialdehyde production occurred at relatively high subzero temperatures. This peroxidation was severe and progressive in the - 20°C group. These studies agree with examples of lipid peroxidation known to occur in frozen food substances at relatively high subzero temperatures (12). If tissue specimens are to be stored for batched assay, it must be emphasized that degeneration will occur at temperatures in the -20°C range which may interfere with the assay. This becomes important in any assay of free radical activity. The lipid peroxidation is suppressed by low subzero temperatures and we therefore recommend that specimens for batched assay be stored in the - 196°C range. The glutathione .ratio and the level of conjugated dienes are seen to rise with the period of tissue storage. This again was marked at - 20°C. Even in those samples stored at - 196°C by Day 1 there were notable rises in glutathione ratios and conjugated dienes, which then did not change with longer storage. It is difficult to believe that peroxidative changes could occur at
AND HOBBS
- 196°C. However, the changes in glutathione ratios and dienes could represent early changes during the freezing or thawing process, which were stabilized once the samples had reached very low temperatures. Conjugated diene formation is thought to occur at an earlier stage in the lipid peroxidation pathway and would therefore be expected to rise at an earlier stage in the storage period. Glutathione in its reduced form acts as a defense mechanism within cells subjected to oxidative stress. The rise in glutathione ratio represents an increase in the oxidation of glutathione and subsequent fall in reduced glutathione due to free radical activity within the system. Again an earlier rise than that of malondialdehyde production would be expected. The superoxide radical has been implicated in many free radical-mediated processes. In order to demonstrate that this radical is involved in the free radical activity associated with storage of liver homogenate at subzero temperatures, a speciIic deactivator of the superoxide radical was added. Superoxide dismutase is a naturally occurring free radical scavenger and it was demonstrated that the addition of exogenous superoxide dismutase to the homogenate significantly reduced the production of malondialdehyde. The superoxide radical is therefore likely to be involved in this lipid peroxidation reaction. The addition of superoxide dismutase did not reduce the rise in glutathione ratio or the level of conjugated dienes. Free radical activity was sufficient to cause glutathione conversion and to produce conjugated dienes. This rise of the earlier markers of lipid peroxidation may indicate the presence of radicals other than superoxide which would obviously not be deactivated by superoxide dismutase. This is supported by the observation that superoxide dismutase did not totally abolish malondialdehyde production even though it was significantly reduced. These findings may also be explained if the superoxide dismutase could not gain ac-
COLD-STORED
TISSUE DETERIORATION
cess to all areas in the frozen homogenate where peroxidation was proceeding. The reduction of lipid peroxidation may also be dose dependent and in this experiment was not fully suppressed by the dose used. Sucrose/EDTA is a combination of a sugar and a chelating agent. It is less specific in its action than superoxide dismutase and as a consequence will scavenge more free radical species. This may explain the complete suppression of malondialdehyde production at -20°C and the effects upon conjugated dienes and glutathione ratio at 28 days. The role of transition metals in free radical activity and lipid peroxidation has been described previously (7). The presence of a metal chelator (EDTA) in the storage solution may well have a beneficial effect by lim iting some of the metal catalyzed steps in the lipid peroxidation pathway. One interesting aspect of the work is that the addition of antiradical agents (SOD and sucrose/EDTA) altered some aspects of oxidative stress. Free radical damage has been implicated as one of the causes of loss of viability of cells during or just after freezing. Previously there have been some studies on the use of antioxidants in cell cryopreservation (9), and our results suggest that this m ight be a valuable area for further study, particularly to avoid oxidative damage as cells pass through conditions of high subzero temperatures during cooling or rewarming.
DUE TO LIPID PEROXIDATION
4.
5.
6.
7.
8. 9.
10.
11.
12.
REFERENCES 1. Adkison D., Hallwarth M. E., Benoit, J. N., Parks, D. A., McCord, J. M., and Granger, D. N. Role of free radicals in ischaemiareperfusion injury to the liver. Acta Physiol. Stand. Sup& 548, 101-107 (1986). 2. Bird, R. P., and Draper, H. H. Comparative studies on different methods of malondialdehyde determination. Merhods Enzymol. 105, 299-30.5 (1984). 3. DeGroot, H., and Noll, T. Haloalkane free radi-
13.
14.
673
cals and lipid peroxidation under low steady state oxygen partial pressures. Biochem. Pharmacol. 35(l), 15-19 (1986). Eklow-Lastbom, L., Rossi, L., Thor, H., and Orrenius, S. Effects of oxidative stress caused by hyperoxia and diquat: A study in isolated hepatocytes. Free Rad. Res. Commun. 2(2), 57-68 (1986). Esterbauer, H., Cheeseman, K. H., Dianzani, M. U., Poli, G., and Slater T. F. Separation and characterisation of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2 + in rat liver microsomes. Biochem. J. 208(l), 129-140 (1982). Hissin, P. J., and Hilf, R. Fluorimetric method for the determination of oxidised and reduced glutathione in tissues. Anal. Biochem. 74, 214-226 (1976). Horton, R., Rice-Evans, C., and Fuller, B. The effects of iron mediated oxidative stress in isolated renal cortical brush border membrane vesicles at normothermic and hypothermic temperatures. Free Rad. Res. Commun. 5, 267-275 (1989). Kappus, H., In “Oxidative Stress” (H. Sies, Ed.). Academic Press, London, 1985. Killian, G., Honadel, T., McNutt, T., Henault, M., Wegner, C., and Dunlap, D. Evaluation of butylated hydroxytoluene as a cryopreservative added to whole or skim milk diluent for bull semen. J. Dairy Sci. 72, 1291-1295, (1989). Reynolds, E. S., and Moslen, M. T., Free radical damage in liver. In “Free Radicals in Biology” (W. A. Pryor, Ed.), p. 49. Academic Press, London, 1980. Slater, T. F., Overview of methods used for detecting lipid peroxidation. Methods Enzymol 105, 283-293, (1984). Tomas, M. C., and Anon, M. C. Study on the influence of freezing rate on lipid oxidation in fish (salmon) and chicken breast muscles. Int. J. Food. Sci. Technol. 25, 718-721, (1990). Wong, S. H. Y., Knight, J. A., Hopfer, S. M., Zacharia, O., Leach, C. N., and Sunderman, F. W. Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin. Chem. 33, 21&220, (1987). Yagi, K. Assay for serum lipid peroxide level and its clinical significance. In “Lipid Peroxides in Biology and Medicine,” p. 233. Academic Press, San Diego, 1982.
29, 668-673 (1992)
Deterioration of Cold-Stored Tissue Specimens Due to Lipid Peroxidation: Modulation by Antioxidants at High Subzero Temperatures G . S. W. WHITELEY, University Department
B. J. FULLER, AND K. E. F. HOBBS
of Surgery, Royal Free Hospital and School of Medicine, Hampstead, London NW3 2QG, United Kingdom
Pond Street,
It is often necessary to store tissue specimens in subzero conditions for assay in batches. During storage at -20°C we found that sufftcient lipid peroxidation occurred in rat liver homogenates in phosphate-buffered saline to affect subsequent malondialdehyde assays. This peroxidation did not occur at - 196°C. The ratio of oxidized to reduced glutathione increased with storage at - 20°C and the level of conjugated dienes increased progressively. The addition of a specific free radical scavenger, superoxide dismutase (200 u/ml) reduced the level of malondialdehyde (P < 0.001) during -20°C storage for periods of 28 days but failed to prevent the changes in the glutathione ratio or dienes. Storage in a less specific free radical scavenger, 0.25 molar sucrose/EDTA, instead of phosphatebuffered saline totally prevented the malondialdehyde production over similar storage periods. o 1992 Academic Press, Inc.
Lipid peroxidation due to the action of oxygen-derived free radical species on polyunsaturated fatty acids has been reported in liver tissue during unfavorable conditions. Insults including ischemia and reperfusion injury (1, 3) and the administration of toxins (4, 11) will lead to lipid peroxidation. Freezing is an insult which combines cellular disruption with chemical change. In many biochemical or pharmacological studies, liver samples or homogenates are frozen for subsequent assay. Since mammalian liver cell membranes are rich in polyunsaturated acids and the products of lipid peroxidation have many pharmacological actions by themselves, it is of interest to consider the possibility that freeze-storage of liver samples may lead to lipid peroxidation. Malondialdehyde is an end product of lipid peroxidation (5, 8) which can be measured by several techniques (2, 11, 13, 14). Conjugated dienes, produced at an early stage in the lipid peroxidation pathway to Received August 14, 1991; accepted July 3, 1992
malondialdehyde, can also be measured. The oxidation of reduced glutathione to oxidized glutathione is an important endogenous defense mechanism against free radical activity. The measurement of both oxidized and reduced glutathione acts as a third measure of free radical activity. This study shows that measurable lipid peroxidation takes place in frozen tissues which leads to tissue deterioration. More interestingly, the addition of a specific scavenging enzyme, superoxide dismutase, during the freezing period altered some aspects of this oxidative damage, while a more general scavenger, sucrose/EDTA, containing a sugar together with a chelating agent had a greater effect. MATERIALS
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS
The experiments used three groups of 10 male Sprague-Dawley rats weighing between 200 and 250 g. The rats were anesthetized using ether inhalation and hepatectomy was performed. Homogenization was performed as rapidly as possible in iced Dulbecco’s A phosphate-buffered saline
668 OOll-2240/92 $5.00
AND
COLD-STORED
TISSUE
DETERIORATION
(PBS) using a rotary PTFE plunger and glass tube. Homogenate (5 ml) samples were set aside as the control (control group A) for each test and an immediate assay for malondialdehyde (MDA) using a thiobarbituric acid reaction (14) was carried out on 10 specimens to act as a control. Simultaneous assays were performed for oxidized and reduced glutathione together with an assay for conjugated dienes. Homogenate was quick-frozen by rapid transition through nitrogen vapor into liquid nitrogen (liquid nitrogen storage group C) or frozen in a freezing cabinet at - 20°C ( - 20°C storage group B). Storage periods of 1, 7, and 28 days were studied. The assays were performed after the specified storage periods on 10 specimens in each experimental group. Samples of liver homogenate to which 200 u/ml of superoxide dismutase (from bovine erythrocytes) was added were stored concurrently at both temperatures (liquid nitrogen storage + SOD group D; -20°C storage + SOD group E). These samples were compared with the untreated homogenate. A further group of samples which had been homogenized in 0.25 molar sucrose/ EDTA instead of Dulbecco’s phosphatebuffered saline was concurrently stored at both temperatures (liquid nitrogen storage + sucrose/EDTA group F; -20°C storage + sucrose/EDTA group G). They were assayed after the same storage periods. A summary of the experimental groups follows: Group A, Control, freshly prepared homogenate in PBS. Group B, PBS homogenate frozen to -20°C and then stored. Group C, PBS homogenate frozen to - 196°C and then stored. Group D, - 196°C storage + SOD. Group E, -20°C storage + SOD. Group F, -’ 196°C storage + sucrose/ EDTA. Group G, -20°C storage + sucrose/ EDTA.
DUE TO LIPID
PEROXIDATION
669
Assay Methods
Prior to assay, all specimens were quickthawed in a 37°C water bath. The homogenates were mixed thoroughly on a rotary shaker and samples were immediately transferred to the assay reaction vials. Malondialdehyde was assayed using a thiobarbituric acid reaction (14) in duplicate lo-ml plain tubes with screw caps. Each tube contained 0.2 ml 7% sodium lauryl sulfate, 2.0 ml 0.1 molar hydrochloric acid, 0.3 ml 10% phosphotungstic acid, and 1.0 ml 0.67% thiobarbituric acid. Two milliliters of homogenate sample was added to each tube. A 16-nmol malondialdehyde standard was set up together with a reagent blank using 2 ml of Dulbecco’s A phosphatebuffered saline. The contents of the tubes were mixed and the tubes were incubated at 95°C for PBS samples and 85°C for sucrose samples (to prevent caramelization of the sucrose) in a water bath for 1 h. At the end of the incubation the tubes were placed on ice for 10 min. Ice-cold butan-l-01 (5 ml) was added. The tubes were well mixed and centrifuged at 3000 rpm for 10 min. The upper butan-l-01 layer was removed and read on a Perkin-Elmer LS5 fluorimeter using a 515nm excitation and a scanning emission between 540 and 560 nm. Conjugated dienes were estimated using extraction by 8 ml of a 2:l chloroform: methanol mixture to which 400 ~1 of liver homogenate had been added. Thorough mixing was performed followed by centrifugation at 2500 r-pm for 5 min. A blank using 400 ~1 of Dulbecco’s phosphate-buffered saline was set up. The samples were duplicated and read against the blank on an ultraviolet spectrophotometer at a wavelength of 240 nm. The reduced glutathione assay (6) was performed as follows. A 5O-kl sample of liver homogenate was diluted with 450 t.~lof phosphate EDTA buffer at pH 8. A IOO-~1 sample of this initial dilution was added to
670
WHITELEY,
FULLER,
3.7 ml of phosphate EDTA buffer in a lo-ml plain glass blood tube. To this mixture 200 ~1 of orthophthalaldehyde (0.02 g in 20 ml methanol) was and the contents were mixed. The tubes were incubated at room temperature for 15 min. All samples were assayed in duplicate against a standard and phosphate EDTA blank on a Perkin-Elmer LS5 fluorimeter at an excitation of 350 nm and a scanning emission between 410 and 440 nm. The oxidized glutathione (6) was assayed as follows. A 500-~1 sample of liver homogenate was mixed with 200 ~1 of 0.04 molar n-ethyl maleimide which had been freshly prepared. This mixture was incubated at room temperature for 30 min. To quench the reaction and bring the pH to 12, 4.3 ml of 0.1 M sodium hydroxide was added at the end of this period. An aliquot of 100 ~1 was taken and diluted with 3.7 ml of 0.1 M sodium hydroxide. Two hundred microliters of orthophthalaldehyde (0.02 g in 20 ml methanol) was added and the mixture incubated at room temperature for 15 min. The samples were read in duplicate against a standard and reagent blank at an excitation of 350 nm and a scanning emission between 410 and 440 nm. The malondialdehyde concentration was calculated in nmol/g liver. The glutathione ratio (oxidized/reduced) was calculated and the level of conjugated dienes were measured in fluorescence units per gram liver. Statistical analysis between groups was
AND HOBBS
performed using an unpaired Student t test. A statistical level of P < 0.01 or better was accepted for significance. RESULTS
The data for liver homogenate stored at - 20°C (group B) and - 196°C (group C) are shown in Table 1. The effect of storage temperature on malondialdehyde is shown in Fig. 1. The effects of superoxide dismutase (groups D and E) and sucrose/EDTA (groups F and G) on malondialdehyde production are shown in Fig. 1. A malondialdehyde assay was performed after 2 h freezing at both - 20 and - 196°C on 10 PBS homogenate samples to assess the effect of the freeze-thaw cycle. There was no significant difference in malondialdehyde production over control. Table 2 shows values for all measurements of peroxidation for all experimental groups at 1 and 28 days of storage. Malondialdehyde production was shown to occur in the liver tissue stored at - 20°C (group B) and was greater than the control (group A) level after only 24 h storage (P < 0.001). The level of malondialdehyde rose after 7 and 28 days storage to a significant level (P < 0.001). The level of malondialdehyde reached in the -20°C (group B) storage group was very high, reaching approximately five times the control level at 28 days. However, in the group stored at - 196°C (group C) the production of malondialdehyde in liver homogenates was suppressed.
TABLE 1 Values for MDA Content, Oxidized/Reduced Glutathione Ratio, and Conjugated Dienes in Liver Homogenates in PBS at -20°C (Group B) and - 196°C (Group C) during 28 Days Storage (means 2 SD)
Group B Day Day Day Day
0 1 7 28
81.1 178.2 337.4 445.8
+ 2 k 2
17.3 31.9* 31.9* 38.6*
* P < 0.001 from control.
Group C 81.1 68.0 66.0 93.3
k 2 2 2
Conjugated dienes fluorescence units/g
Oxidized/reduced glutathione ratio
MDA content (nmoYg)
17.3 16.6 13.3 12.0
Group B 0.41 1.09 1.69 1.43
2 ” 2 5
0.04 0.38* 0.22* 0.07*
Group C 0.41 0.94 0.99 1.08
f 2 2 ”
0.04 0.03* 0.10* 0.17*
Group B 6.8 7.7 8.7 10.4
* 2 + 2
0.4 0.8 0.9 0.6
Group C 6.8 8.6 8.5 8.1
f k * t
0.4 0.2 0.6 0.3
COLD-STORED
0
TISSUE
DETERIORATION
7
StoragePerti khys) .
FIG. 1. The effect of storage temperature on malondialdehyde production (nmol/g) in stored liver homogenate (mean + SD, n = 10).
Superoxide dismutase added prior to freezing reduced the level of malondialdehyde over control (P < 0.001) at 1,7, and 28 days storage at -20°C (group D). Nevertheless, superoxide dismutase did not totally prevent the rise in malondialdehyde level (Fig. 2). Malondialdehyde production was reduced by storage in 0.25 M sucrose/ EDTA (group F). There was no significant rise in malondialdehyde production at 1, 7, and 28 days storage at - 20°C over the control group.
DUE TO LIPID
PEROXIDATION
671
The glutathione ratio (oxidized/reduced) increased with the period of storage. The ratio reached a significant (P < 0.001) level at -20°C (group B) after 1 day. The values at -20°C were higher than those stored at - 196°C (groups C vs B) (P < 0.001) at all storage intervals. However, superoxide dismutase did not prevent the rise in glutathione ratio with increasing storage periods at - 20°C. In the group stored at - 20°C in sucrose/EDTA the glutathione ratio was no different than the group stored without antioxidant (groups F vs B). Conjugated diene level rose with the duration of storage at -20°C (group B) achieving a significantly high level at 28 days (P < 0.001). At - 196°C (group C) there was no significant increase in the level of conjugated dienes throughout the period of storage. Superoxide dismutase failed to prevent the rise in diene level in group B at -20°C. Sucrose/EDTA storage at -20°C (group F) reduced the level of conjugated dienes at 28 days (P < 0.001). The combined changes in MDA content, oxidized/reduced glutathione ratio, and conjugated diene levels in the experimental groups on Day 1 and Day 28 are shown in Table 2.
TABLE
DISCUSSION
From the above experiment we have 2
Values for MDA Content, Oxidized/Reduced Glutathione Ratio, and Conjugated Dienes (means f SD) in Liver Homogenates after 1 and 28 Days Storage MDA content (nmol/g)
Group B ( - 20°C storage) Group C (- 196°C storage) Group D (- 196°C + SOD) Group E (-20°C + SOD) Group F f - 196°C f SucroselEDTA) Group G ( - 20°C + Sucrose/EDTA) * P < 0.001 from control.
Oxidized/reduced glutathione ratio
Conjugated dienes (fluorescence/g)
Day 1
Day 28
Day 1
Day 28
Day 1
Day 28
178.2 + 31.9*
445.8 2 38.6*
1.09 + 0.38*
1.43 + 0.07’
7.7 + 0.8
10.4 + 0.6
68.0 k 16.6
93.3 2 12.0
0.94 i 0.03*
1.08 + 0.17*
8.6 ? 0.2
8.1 + 0.3
70.11 + 4.15
85.03 2 3.90
0.91 2 0.10*
1.51 2 0.07*
6.9 2 0.1
7.5 It 0.5
117.82 i 5.89*
370.46 ? 1I .05*
1.20 k 0.04*
2.01 5 0.12*
6.4 2 0.6
8.4 k 1.6
65.50 t 6.97
104.02 5 7.28
1.09 -c 0.05*
1.75 -c 0.05*
6.4 f 0.3
6.5 k 0.4
66.97 + 3.23
113.44 f 6.68
1.07 f 0.03;
1.60 k 0.06*
5.9 t 0.6
5.6 + 0.5
672
WHITELEY,
FULLER,
PBSalone(GroupB ) PBSt 200~I ml SODI GroupE 1 PBSt 0.25MSucroselEDTAl GroupG I
StoragePeriod(days)
FIG. 2. The effect of superoxide dismutase and sucrose/EDTA on malondialdehyde production (nmoVg) in liver homogenate stored at - 20°C (mean 2 SD, n = 10).
demonstrated that lipid peroxidation of liver homogenates as measured by malondialdehyde production occurred at relatively high subzero temperatures. This peroxidation was severe and progressive in the - 20°C group. These studies agree with examples of lipid peroxidation known to occur in frozen food substances at relatively high subzero temperatures (12). If tissue specimens are to be stored for batched assay, it must be emphasized that degeneration will occur at temperatures in the -20°C range which may interfere with the assay. This becomes important in any assay of free radical activity. The lipid peroxidation is suppressed by low subzero temperatures and we therefore recommend that specimens for batched assay be stored in the - 196°C range. The glutathione .ratio and the level of conjugated dienes are seen to rise with the period of tissue storage. This again was marked at - 20°C. Even in those samples stored at - 196°C by Day 1 there were notable rises in glutathione ratios and conjugated dienes, which then did not change with longer storage. It is difficult to believe that peroxidative changes could occur at
AND HOBBS
- 196°C. However, the changes in glutathione ratios and dienes could represent early changes during the freezing or thawing process, which were stabilized once the samples had reached very low temperatures. Conjugated diene formation is thought to occur at an earlier stage in the lipid peroxidation pathway and would therefore be expected to rise at an earlier stage in the storage period. Glutathione in its reduced form acts as a defense mechanism within cells subjected to oxidative stress. The rise in glutathione ratio represents an increase in the oxidation of glutathione and subsequent fall in reduced glutathione due to free radical activity within the system. Again an earlier rise than that of malondialdehyde production would be expected. The superoxide radical has been implicated in many free radical-mediated processes. In order to demonstrate that this radical is involved in the free radical activity associated with storage of liver homogenate at subzero temperatures, a speciIic deactivator of the superoxide radical was added. Superoxide dismutase is a naturally occurring free radical scavenger and it was demonstrated that the addition of exogenous superoxide dismutase to the homogenate significantly reduced the production of malondialdehyde. The superoxide radical is therefore likely to be involved in this lipid peroxidation reaction. The addition of superoxide dismutase did not reduce the rise in glutathione ratio or the level of conjugated dienes. Free radical activity was sufficient to cause glutathione conversion and to produce conjugated dienes. This rise of the earlier markers of lipid peroxidation may indicate the presence of radicals other than superoxide which would obviously not be deactivated by superoxide dismutase. This is supported by the observation that superoxide dismutase did not totally abolish malondialdehyde production even though it was significantly reduced. These findings may also be explained if the superoxide dismutase could not gain ac-
COLD-STORED
TISSUE DETERIORATION
cess to all areas in the frozen homogenate where peroxidation was proceeding. The reduction of lipid peroxidation may also be dose dependent and in this experiment was not fully suppressed by the dose used. Sucrose/EDTA is a combination of a sugar and a chelating agent. It is less specific in its action than superoxide dismutase and as a consequence will scavenge more free radical species. This may explain the complete suppression of malondialdehyde production at -20°C and the effects upon conjugated dienes and glutathione ratio at 28 days. The role of transition metals in free radical activity and lipid peroxidation has been described previously (7). The presence of a metal chelator (EDTA) in the storage solution may well have a beneficial effect by lim iting some of the metal catalyzed steps in the lipid peroxidation pathway. One interesting aspect of the work is that the addition of antiradical agents (SOD and sucrose/EDTA) altered some aspects of oxidative stress. Free radical damage has been implicated as one of the causes of loss of viability of cells during or just after freezing. Previously there have been some studies on the use of antioxidants in cell cryopreservation (9), and our results suggest that this m ight be a valuable area for further study, particularly to avoid oxidative damage as cells pass through conditions of high subzero temperatures during cooling or rewarming.
DUE TO LIPID PEROXIDATION
4.
5.
6.
7.
8. 9.
10.
11.
12.
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