Lipid peroxidation in fish muscle microsomes in the frozen state

CRYOBIOLOGY

Lipid

19, 154- 162 (1982)

Peroxidation

in Fish Muscle

MARTIN

E. APGARl

Massachusetts Agricultural Experiment Station, Laboratory, University of Massachusetts

Microsomes HERBERT

AND

EXPERIMENTAL

Isolation of microsomal fraction. Live winter flounder (Pseudopleuronectes americanus) were sacrificed by decapitation and immediately filleted. The fillets were minced in a Rival meat grinder. Four volumes of buffer (0.12 M KCl, 5 n-u%4histidine, pH 7.3) was added to 45-g samples of minced muscle. A Brinkman polytron was Received July 14, 1981; accepted December 8, 1981 ’ Present address: Hershey Foods Corp., Technical Center, 1025 Reese Ave., Hershey, Pa. 17003.

Copyright AU rights

0 1982 by Academic Ress, Inc. of reproduction in any form reserved.

and Nutrition, Massachusetts

Marine 01930

Foods

used to homogenize the mixture with two 30-set bursts. The homogenate was centrifuged at 21,400g for 30 min, and the sediment was discarded. The supernatant fraction was then centrifuged at 104,OOOg for 60 min followed by resuspension of the sediment in 0.6 M KC1 to solubilize actomyosin. Centrifugation was again carried out at 104,OOOg for 60 min with the sediment being defined as the microsomal fraction. The supematant fraction was discarded and the sediments were taken up in the desired volume of 0.12 M KC1 buffer. The resulting mixture was homogenized using a Potter-Elvehjem-type tissue grinder. The procedure is essentially that of McDonald et aE. (11). Peroxidation assays: 6°C. Assay mixtures were made up with the following concentrations (unless otherwise noted): 0.5 mg of microsomal protein/ml, 0.1 mM NADH, and 0.1 m&f ADP. The reaction was initiated by addition of enough FeCl, to reach a final concentration of 0.01 m&f. Immediately after addition of iron the reaction mixture was swirled, and a l-ml aliquot was removed and added to 2 ml of 20% (w/v) trichloroacetic acid. Thiobarbituric acid-reactive substances were measured as described below. When assay mixtures were prepared in triplicate 3 vol of a microsomal fraction was separately added to three separate solutions containing the desired concentrations of cofactors. Correlation coefficient analyses were used to show differences in the amount of product formed over time between two different treatments ( 13). Subzero. Assay mixtures were made up 154

001 l-2240/82/020154-09$02.00/O

0. HULTIN

Department of Food Science Marine Station, Gloucester,

Lipid oxidation is one of the primary mechanisms of chemical decomposition in stored muscle tissue. Due to its high content of polyunsaturated fatty acids, fish muscle is especially susceptible to this type of deterioration. Fatty-type fish may undergo rapid lipid oxidation during refrigerated storage. This is not usually as significant a problem with non-fatty-type fish since microbial spoilage often occurs before the onset of lipid peroxidation. In frozen lean fish, however, where microbial growth is surpressed, lipid oxidation may contribute to long-term quality changes manifested by changes in flavor, texture, or nutritional quality (6, 9, 18). Although the oxidation of lipids in stored muscle tissue has generally been ascribed to nonenzymic processes, we have recently demonstrated the presence of an enzymic lipid peroxidation system in a microsomal membrane fraction of fish muscle (11). In this study we have characterized the lipid peroxidative system of flounder muscle at temperatures below freezing.

in the Frozen State

LIPID

OXIDATION

IN

with the desired concentration of cofactors. The reaction was initiated by addition of iron. One-milliliter aliquots were withdrawn at once from the reaction mixture and transferred into 25ml Erlenmeyer flasks held on ice. The flasks were immediately transferred to a freezer held at - 90°C. After 5 min they were removed and placed in a freezer held at the desired reaction temperature. After 15 min (to allow temperature equilibration) one flask was removed to which 2 ml of 20% trichloroacetic acid was added. This sample served as the control. Other samples were removed after appropriate time intervals. The samples were allowed to thaw at room temperature. Thiobarbituric acid (TBA)-reactive substances were measured as described below. Values for the control were subtracted from those of the other samples. Measurement of thiobarbituric acidreactive substances. The enzymic reaction was halted by addition of 2 ml of a 20% trichloroacetic acid (TCA) solution to a l-ml aliquot of the assay mixture for lipid oxidation. The mixture was centrifuged for 15 min at approximately 1600g. A 1.5-ml aliquot of the supernatant fraction was transferred to a test tube containing 2.0 ml of aqueous 0.67% TBA. The tubes were heated in a boiling-water bath for 15 min. Optical density was measured at 532 nm and results are reported as nanomoles of malondialdehyde (MDA) per milligram of protein using the extinction coefficient EgZm = 1.56 >: 105M-’ (2). For the remainder of this paper all references toward a rate of reaction are: referring to the enzymic lipid oxidation reaction unless otherwise specified. Protein measurement. The protein concentration of the microsomal suspension was determined using the modified Lowry procedure of Markwell et al. (10). Separation of temperature and freezing effects. Four different buffer solutions were prepared that prevented freezing at - 12°C. All contained 0.12 M KCl, 5 mM histidine

FROZEN

MICROSOMES

155

(pH 7.3). Two solutions contained methanol at 20 and 30% (v/v), where the other two solutions contained ethanol at the same two volume fractions. Subzero assays were carried out in the same manner as for aqueous buffer solutions. Lipid hydroperoxide determination. The method of Buege and Aust (3) was used. Five milliliters of chloroform:methanol (2:l) was added to a l-ml aliquot of the assay mixture for lipid oxidation. This mixture was centrifuged at approximately 16OOg for 5 min. Most of the upper phase was removed by suction. Three milliliters of the lower phase was removed by syringe and transferred to a test tube which was held in a 45°C water bath. The tube was taken to dryness under a stream of nitrogen. While remaining under nitrogen 1 ml of acetic acid:chloroform (3:2, previously bubbled with nitrogen at 4°C) and 0.05 ml of a KI solution (6 g of KI in 5 ml of water) were quickly added. The tubes were stoppered, mixed, and placed in the dark for 5 min. Three milliliters of a cadmium acetate solution (0.5 g in 100 ml H20) was added, the solution mixed, and its absorbance determined at 353 nm. The results are reported as nanomoles of hydroperoxide using the extinction coefficient E1-c& = 1.73 104 M-1. Addition of soluble muscle components. Eighty grams of minced fish muscle was homogenized in a Waring blender in two lo-set bursts. The homogenate was centrifuged at 104,OOOg for 8 hr. The supernatant, defined as the press juice, was recovered and divided into two fractions. One fraction was passed through a PM-10 ultrafiltration membrane (Amicon). The filtrate was designated the low-molecular-weight fraction. The other fraction was dialyzed for 16 hr against a 0.12 M KCl, 5 mM histidine, pH 7.3, solution, with one buffer change after 10 hr. The retentate was designated the high-molecular-weight fraction. Comparison of the effects of the high- and lowmolecular-weight fractions between the two

156

APGAR AND HULTIN

temperatures used (-5°C and +6”C) was done by analysis of variance (13). pH determination in frozen state. Tenmilliliter volumes of 0.12 M KCl, 5 m&I histidine solution was adjusted to pH values of 5.0 to 7.5 at 0.5-unit intervals. To these solutions p-nitrophenol was added to a concentration of 0.05 m&I. Separate KCl-histidine solutions were adjusted to pH values of 6.5 to 8.0 at 0.5-unit intervals; m-nitrophenol was added to a concentration of 0.15 n&f. These were the standard tubes. Experimental duplicate tubes contained 0.15 M NaCl or 0.15 M KC1 (both contained 5 mM histidine, pH 7.3). m-Nitrophenol was added to one pair of duplicate tubes, whilep-nitrophenol was added to the other. Both the standard and experimental tubes were held at -90°C for 20 min and then transferred to a freezer at -5°C for 30 min. Experimental tubes containing m-nitrophenol were compared with standard tubes containing m-nitrophenol. The same method was used for tubes with p-nitrophenol to obtain pH values of experimental tubes. Comparison of tubes containing NaCl with tubes containing KC1 were made to obtain relative pH values. This is a modification of the method of Fishbein and Winker? (4). RESULTS

The effect of below freezing temperature on the rate of the lipid peroxidation reaction is demonstrated in Fig. 1. For these experiments, assay mixtures were prepared in triplicate for each temperature and the same microsomal preparation was used for all temperatures. The rate of the oxidation decreased with decreasing temperatures. The sample reacting at -20°C appeared to reach a constant level of MDA after approximately 60 min. It is possible this is due to the smaller amount of 0, in the reaction medium at this temperature. The zero time value was obtained by subtracting the amount of malondialdehyde (MDA) after freezing and thawing of the sample. A control which was heated to 100°C for 10 min

60

90

TIME(min)

FIG. 1. Effect of subzero temperature on oxidation. Assay mixtures were made up in triplicate and assayed at -2°C (0, -12°C (O), and -20°C (Cl). The medium contained 0.0075 m&4 FeCl,, 0.1 mM NADH, 0.1 mM ADP, and 0.5 mg/ml of protein. All samples were prepared from the same microsomal preparation, and bars indicate standard deviations.

showed an oxidative rate of less than 10% of that of the unheated sample. The residual peroxidation could be due to either enzyme which was not deactivated by the heat treatment or by nonenzymic oxidations. The rate of enzymic lipid peroxidation was determined at unfrozen and frozen temperatures in the presence of 0.15 M NaCl or 0.15 M KC1 and the usual components of the assay mixture (Fig. 2). At 6°C there was no difference in the rate of MDA formation in the presence of either the Na+ or K+, while at - 12°C the reaction mixture containing NaCl had a higher rate of reaction than the solution containing KCl. A correlation coefficient analysis indicated that this difference was significant (P < 0.005). The effect of increasing concentrations of NaCl or KC1 on the peroxidation rate of the microsomal fraction at 6°C is shown in Fig. 3. There are large decreases with increasing concentrations of both of these salts. This decrease in activity is rapid at first and then levels off. Because of the rather large scatter of the data in these experiments, it was not possible to determine if there was a difference in the effect of the two cations on the peroxidation reaction. With other factors held constant, there

LIPID

OXIDATION

IN

FROZEN

157

MICROSOMES

$ .c $

36-

i

24-

p

18.

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12-

30-

6-

J, TIMEChr)

2. Effect of sodium chloride versus potassium chloride on oxidation. Assay mixtures containing 0.01 mM FeCl,, 0.1 mM NADH, 0.1 mM ADP, 0.5 mg/ml of protein were prepared in triplicate in either 0.15 M NaCl or 0.15 M KC1 (both contained 5 m&I histidine, pH 7.3). Assay mixtures were run in triplicate for sodium containing (- 12°C O--O, 6”C, O---O) and potassium containing (- 12”C, -0, O- - - 0) mixtures. Thick bars indicate standard deviations for sodium containing mixtures, and thin bars indicate standard deviations for potassium containing mixtures. FIG.

was a decrease in specific activity of the microsomal peroxidative activity with increasing concentrations of microsomal protein over the range of 0.1 to 2.0 mg/ml (Fig. 4). These results indicate that something other than protein is rate limiting in the

[SALT], M 3. Effect of salt concentration on oxidation. Assay mixtures were prepared in duplicate containing varying concentr,ations of either NaCl (0) or KC1 (0) with all solutions buffered with 5 mM histidine at pH 7.3. Cofactors were 0.01 mM FeCl,, 0.1 mM NADH, 0.1 nut4 ADP, and 0.5 mg/ml of protein. Incubation was at 6°C. FIG.

,

O 0.10cl25

0.50 [IR~TEI

1.0

2.0

Nl, mg/ml

FIG. 4. Effect of protein concentration on oxidation in frozen state. Protein concentration was varied over the range 0.1 to 2.0 mg/ml while the concentrations of oxidative cofactors were held constant at 0.1 mM NADH, 0.1 mM ADP, and 0.01 mM FeCl,. Specific activities were calculated based on 60-min readings. The assays were run in duplicate at - 12°C.

system. One possibility is that the low amount of unfrozen water can hold only a limited amount of 0, which becomes rate limiting. Another possibility is that either the protein or the phospholipid of the membrane is chelating Fe thus lowering the amount of free Fe and limiting the reaction. A further possibility is that the microsomal fraction contains an inhibitor which is solubilized in the assay medium or in the last preparative step. Increasing the amount of microsomes would increase the concentration of the inhibitor in the assay medium. Lipid hydroperoxides and their breakdown products are formed at different steps of the oxidative reaction. In two different experiments, the relative rate of formation of hydroperoxides and MDA were measured as a function of temperature (below freezing) (Table 1). The ratio of lipid hydroperoxide to MDA is relatively constant throughout both sets of experiments over the range of temperature examined. These data are consistent with the hypothesis that the rate-determining step for the reaction occurs before hydroperoxide formation. We assessed the contribution of ice crystal formation in the reaction by comparing the lipid peroxidation rate of the

158

APGAR

Effect

of Temperature Hydroperoxide

Temperature (“C)

nmol MDA mg protein

-2 -12 -20 -5 -13 -22 N&e.

TABLE 1 on Relative and MDA nmol

mixtures

of Lipid

LOOH

nmol

MDA

mg protein

nmol LOOH

107 96.7 32.7 125 80.2 37.6

0.147 0.110 0.147 0.106 0.150 0.091

15.8 10.6 4.79 13.2 12.0 3.41 Assay

Amount Formed

in triplicate

AND

contained and

Fe& 0.1 mM NADH, 0.1 mM ADP, protein. The time of assay was 60 min.

0.5

0.0075 mu mglml of

microsomes in frozen and unfrozen systems at the same subzero temperature. This was accomplished by the addition of methanol or ethanol to the KCl-histidine solution. The oxidation reaction rate in the presence of alcohol could not be directly compared with the oxidation system without the alcohol because both methanol and ethanol were found to inhibit the reaction in the temperature range above 0°C. Thus, an indirect comparison was made by comparing the ratio of the reaction rate at 6°C to that at - 12°C for the systems containing alcohols and the system without the alcohol (Table 2). The ratio for the reaction rate at 6°C compared to -12°C was higher for the assay mixtures containing alcohol than for the assay mixtures without it, i.e., the

Comparison

of the Relative

HULTIN

reaction rate decreased less in the presence of ice than in the presence of alcohols. These results are consistent with an increase of the reaction rate in the presence of ice crystal formation. We believe that this difference is not due to the freezing process itself since microsomes which were subjected to a freeze-thaw cycle and then assayed at above freezing temperatures had exactly the same rate of lipid peroxidation as those which were not frozen and thawed. The high- (> 10,000) and low- (
TABLE 2 Reaction Rates of Frozen nmol

and Unfrozen

MDA nmol

mg protein

Solvent

Mixtures

nmol

MDA MDA

6°C

- 12°C

Methanol 20% (v/v) 30% (v/v)

19.4 22.4

6.98 6.02

2.78 3.12

Ethanol 20% (v/v) 30% (v/v)

24.3 17.7

7.44 6.46

3.27 2.74

Hz0

43.4

20.8

2.09

Note. Solutions were prepared containing dine, 0.12 M KCl, pH 7.3, 0.01 mb4 FeCI,, rates were calculated after 2 hr. Experiments

(6°C) (- 12°C)

0, 20, or 30% (v/v) methanol or ethanol (all contained 5 mM histi0.1 n&f NADH, 0.1 mM ADP, and 0.5 mg/ml of protein). Reaction were performed in triplicate.

LIPID OXIDATION

0.11 ~&OTEI

0.28 t$mg/ml

0.55 (HIGH

1.1 Mw

2.8

IN FROZEN MICROSOMES

5.5

FRACTION)

FIG. 5. Effect of soluble muscle components on oxidation. High- and low-molecular-weight compounds from press juice were added in volumes from 0.01 to 0.50 ml (concentrations kept equal to original press juice) to a final volume of 5 ml, 0.01 m&4 in FeCl,, 0.1 mM in NADH, 0.1 mM in ADP, and 0.5 mg/ml of protein. The corresponding concentrations of protein in the high-molecular-weight fraction are indicated on the abscissa. Samples were taken after 60 min and assayed for MDA. High-molecular-weight (-5°C -0; 6°C O---O), and low-molecularweight (-5°C 0-O; 6°C O---O) compounds. Control samples contained neither high- nor lowmolecular-weight compounds.

fraction functioned more efficiently as an inhibitor at 6 compared to -5°C (P < 0.05). With the low-molecular-weight fraction it was the opposite; there was more inhibition with the latter at -5 compared to 6°C (ZJ < 0.05). DISCUSSION

The data reported in this paper show that the peroxidation of the lipids of the microsomal membrane of winter flounder skeletal muscle proceeds at a highly significant rate in the frozen state, and the rate is temperature dependent. In the frozen state the reaction goes at a faster rate in the presence of 0.15 M NaCl as compared to the same concentration of KCl, a difference which is not observed at 6°C. It has been observed that phosphate-buffered sodium salt solutions become acidic upon freezing and lead to enzyme inactivation, whereas potassium salt causes no change in pH and no enzyme

159

inactivation upon freezing (4, 17). This prompted us to measure the relative pH of the frozen oxidation assay mixtures containing the sodium or potassium. No difference in pH could be observed. Due to the estimated accuracy of this method (20.5 pH unit), the assay mixtures containing sodium or potassium are probably within 1 pH unit of each other. Although a large pH difference does not exist, a difference of less than 1 pH unit could still be responsible for the relative activities of sodium and potassium containing assay mixtures at subzero temperatures. Sussman and Chin (14) showed that when cod muscle was frozen to -12°C approximately 90% of the water in the tissue was frozen. Assuming this value there should be in our system approximately a lO-fold increase in the concentration of solutes. Thus, in theory the concentration of the KC1 and NaCl should be approximately 1 M. KC1 at a concentration of 0.6 M has been shown to inhibit some 35% of the rate of lipid oxidation in a microsomal fraction of chicken muscle (12). We were not able to observe a differpncp in the effect of potassium chloride versus sodium chloride on inhibition of the microsomal lipid peroxidation at temperatures above freezing. It is possible, however, that under conditions of our experiments either a potassium or sodium salt was concentrated to a point past its solubility and precipitated out of the frozen solution. The observed difference in effect of sodium and potassium might possibly be due to preferential removal of one of the ions (presumably Na+) in the frozen reaction mixture. Another possibility is that the differential response of the lipid peroxidation system to sodium and potassium in the frozen state could be related to water structure since sodium is a structure former and potassium is a structure breaker (5). Lipid oxidation in a mitochondrial membrane fraction has been correlated with ionic properties, where oxygen uptake was shown to be in-

160

APGAR

AND

versely proportional to the structure breaking power of an added solute (7). Water-protein interaction has been shown to affect protein conformation (15). Concurrent determination of TBAreactive substances (MDA) and lipid hydroperoxides indicated a constant ratio of these products of the oxidation reaction. In these experiments it is important to consider the possibility that the procedure for the TBA-reactive substances may be detecting hydroperoxides as MDA. When an aliquot is drawn from the oxidative assay mixture and is added to 20% TCA, the enzyme reaction is stopped. The resulting mixture then contains hydroperoxides as well as hydroperoxide breakdown products. Following the addition of a 0.67% thiobarbituric acid solution, the tubes are heated in a boiling-water bath for 15 min. It has been observed that 5% of lipid hydroperoxide breaks down to TBA-reactive substances when heated in a solution containing glycine-HCl (pH 3.6) and 0.293 m&4 FeCl, to 100°C for 15 min (1). Replacement of the glycine-HCI buffer with 35% TCA decreased the amount of hydroperoxide detected as MDA by about 90%. In the present work, TBA-reactive substances were present at a lo-fold lower concentration than lipid hydroperoxides. It seems highly doubtful that the combination of the 200-fold lower concentration of FeCl, (1.43 @Z) and the presence of an inhibitory condition with respect to hydroperoxide breakdown (20% TCA) could allow roughly the same degree of hydroperoxide breakdown that was observed by Asakawa and Matsushita ( 1). Thus, lipid hydroperoxide breakdown was probably insignificant after the enzyme reaction was stopped by addition of the 20% TCA. Tong and Pincock (16) found the reaction rate ratio of frozen to supercooled mixtures was larger than one for invertase-catalyzed hydrolysis of sucrose. This freeze-activation was attributed to the increased concentration of solutes in the liquid regions

HULTIN

of the frozen solutions as compared to the unfrozen solution. Freeze concentration of solutes leading to changes in enzyme activity has been discussed (15). Hazelwood and Dawson (8) observed a maximal rate of phospholipase activity at - 10°C (the temperature at which ice formation was also observed) when activity was measured over the temperature range of -60 to +5O”C. Freeze concentration of solutes may be the reason for the increased rate of reaction in the present system. Since presumably the enzyme and lipids are part of the same subcellular organelle, freezing is probably increasing the effective concentration of components other than the lipid substrate. A reasonable expectation would be that it is the concentration of FeCl, which is involved since we have demonstrated that higher concentrations of NADH and ADP are inhibitory (11). The increased concentration of the FeCl, was overcoming any inhibitory effect of the increased concentration of NADH and ADP. The data on the inhibition of the lipid peroxidation by the soluble components of the muscle must be considered when this system is examined for its effect on lipid oxidation of fish in situ. The relatively greater inhibitory effect of the lowmolecular-weight fraction at - 5°C compared to the unfrozen system could be explained by the increased concentration of the inhibitor due to ice formation. Why the high-molecular-weight fraction inhibits more efficiently at +6”C compared to the frozen system could be due to a diffusional phenomenon. Freezing of the system could limit the enzyme- inhibitor interaction because it inhibits the extent of the diffusion of the high-molecular-weight compound(s) in the frozen system. It is also possible that the high-molecular-weight inhibitors are precipitated out of solution in the frozen state or that either the low temperature or the concentration of solutes at the frozen temperature causes conformational changes in the high-molecular-weight com-

LIPID OXIDATION

ponents, making itors .

IN FROZEN

them less effective inhib-

Graduate School of the University of Massachusetts at Amherst. REFERENCES

SUMMARY

The microsomal fraction from fish muscle has previously been shown to catalyze the oxidation of its lipid. In this study we have studied the rate of the reaction in the frozen state. The rate was dependent on temperature, decreasing with decreasing temperature. When the microsomes were frozen in the presence of NaCl there was greater activity than when they were frozen in the presence of KCl. The specific activity of the oxidation decreased with increasing protein concentration. This is possibly due to the limitation of oxygen in the frozen system. Lipid oxidation is a complex reaction and both initial products (lipid hydroperoxides) and breakdown products (those reacting with malondialdehyde) were measured. This ratio was relatively constant over a variety of conditions indicating that the rate-limiting step of the reaction occurred prior to the formation of lipid hydroperoxide. A study of the reaction at above-freezing temperatures and belowfreezing temperatures in the presence of miscible solvents to prevent freezing at temperatures below 0°C gave results which were consistent with the hypothesis that ice crystal formation had an accelerating effect on the reaction. Presumably this is due to concentration of reactants since freezing and thawing of the microsomes did not affect their rates of lipid oxidation. Potent inhibitors of the lipid oxidation reaction were found in the soluble fraction of the muscle tissue. These were both high-molecular and low-molecular-weight compounds. The low-molecular-weight inhibitors were more effective in the frozen state while the high-molecular-weight compounds were relatively more effective in the reaction catalyzed at temperatures above freezing. ACKNOWLEDGMENTS This work was supported in part by the Massachusetts Agricultural Experiment Station and by the

161

MICROSOMES

1. Asakawa, T., and Matsushita, S. Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides. Lipids 15, 137- 140 (1980). 2. Bidlack, W. R., Okita, R T., and Hochstein, R. The role of NADPH-cytochrome b, reductase in microsomal lipid peroxidation. B&hem. Biophys. Res. Commun. 53, 459-465 (1973). 3. Buege, J. A., and Aust, S. D. Microsomal lipid peroxidation. Methods in Enzymology 52, 302-310 (1978). 4. Fishbein, W. N., and Winkert, S. W. Parameters of freezing damage to enzymes. Advan. Chem. Series

180, 55-82

(1979).

5. Fennema, 0. Water and ice. In “Principles of Food Science, Part I: Food Chemistry” Fennema, O., Ed.), p. 24. Dekker, New York, 1976. 6. Fennema, 0. R., Powrie, W. D., and Marth, E. H. “Low Temperature Preservation of Foods and Living Matter.” Dekker, New York, 1973. 7. Hateti, Y., and Hanstein, W. G. Lipid oxidation in biological membranes I. Lipid oxidation in submitochondrial particles and microsomes induced by chaotropic agents. Arch. Biochem. Biophys.

138, 73-86

(1970).

8. Hazlewood, C. P., and Dawson, R. M. C. A phospholipid-deacylating system of bacteria active in a frozen medium. Biochem. J. 153, 49-53 (1976). 9. Love, R. M. “The Chemical Biology of Fishes. Advances 1968-1977,” Vol. 2, p. 368. Academic Press, New York, 1980. 10. Markwell, M. K., Haas, S. N., Bieber, L. L., and Tolbert, N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 206-210 (1978). 11. McDonald, R. E., Kelleher, S. D., and Hultin, H. 0. Membrane lipid oxidation in a microsomal fraction of red hake muscle. .Z. Food Biochem. 3, 125- 134 (1979). 12. Player, T. J., and Hultin, H. 0. Some characteristics of the NAD(P)H-dependent lipid peroxidation system in the microsomal fraction of chicken breast muscle. J. Food Biochem. 1, 153-171 (1977). 13. Steel, R. G. D., and Torrie, J. H. “Principles and Procedures in Statistics.” McGraw-Hill, New York, 1960. 14. Sussman, M. V., and Chin, L. Liquid water in frozen tissue: Study by nuclear magnetic resonance. Science 151,, 324-325 (1966).

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APGAR AND HULTIN

15. Taborsky, G. Protein alterations at low temperatures: An Overview. Advan. Chem. 180, l-26 (1979). 16. Tong, M. M., and Pincock, R. E. Denaturation and reactivity of invertase in frozen solutions. Biochemistry 8, 908-913 (1969). 17. Van den Berg, L. The effect of addition of sodium and potassium chloride to the reciprocal sys-

tem: KH2P04-Na,HPOsH20 on pH and composition during freezing. Arch. Biochem. Biophys. 84, 305-315 (1959). 18. Weber, F., and Grosch, W. Co-oxydation of a carotenoid by the enzyme lipoxygenase: Influence on the formation of linoleic acid hydroperoxides. Z. Lebensm. Unters. Forsch. 161, 223-230 (1976).