Post-mortem metabolism in fresh porcine, ovine and frozen bovine muscle

Meat Science 19 (1987) 1-14

Post-mortem Metabolism in Fresh Porcine, Ovine and Frozen Bovine Muscle Peter Lundberg*, Hans J. Vogel*t Department of Physical Chemistry 2, University of Lund, 221000 Lund, Sweden

Stefan Fabiansson & H~kan Ruderus Swedish Meat Research Institute, K[ivlinge, Sweden

(Received 16 April 1986; revised version received 10 June 1986; accepted 24 July 1986)

SUMMA R Y Post-mortem metabolism was followed by phosphorus-31-N:14R in muscle samples obtained from freshly slaughtered pigs and lambs. Resonances for creatine phosphate (CP2 ATP, inorganic phosphate (Pi) and sugar phosphates (SP) could be discerned and the intracellular pH could be determined from the spectra. The rates of post-mortem metabolism varied in the following fashion: porcine muscle > ovine muscle > bovine muscle. However, the course of post-mortem metabolism was, in all cases, the same. C P disappeared first and then A TP. Simultaneously, Pi increased, while SP remained relatively constant. The intracellular pH decreased to pH 5"5 in all tissues. In a separate set of experiments the post-mortem metabolism during thawing was studied in bovine muscles that had been frozen immediately after slaughter. Again, the same course of post-mortem metabolism was observed, but the thaw shortening was accompanied by an extremely rapid post-mortem metabolism, which was more than ten times as fast as that measured for fresh bovine muscles. The intracellular pH decreased from 7"2 to 5"5 in 45 min. This rapid metabolism started only after the sample had reached 0° C. Resonances for metabolites were broadened in frozen muscles dtte to the limited motions that are allowed within the ice lattice. * Present address: Division of Biochemistry, Department of Chemistry, University of Calgary, Calgary, Canada. t To whom correspondence should be addressed. I

Meat Science 0309-1740/87/S03"50 ,© Elsevier Applied Science Publishers Ltd. England, 1987. Printed in Great Britain

Peter Lundberg, Hans J. Vogel, Stefan Fabiansson, Hfkan Ruderus

INTRODUCTION Toughness of meat is, to an extent, dependent on the biochemical changes that take place during the first hours post mortem. Normally living muscle cells obtain their biochemical energy (ATP) from respiring mitochondria. This aerobic respiration ceases when the blood circulation stops after slaughter. Under these circumstances the enzymatic machinery of the cell-which is geared to maintaining a constant level of ATP--will initially use creatine phosphate (CP) to generate ATP from ADP. However, the major anaerobic source for post-mortem biochemical energy supply is via the breakdown of glycogen which--through glycolysis--does not only generate ATP but also produces lactate (Ashgar & Pearson, 1980; Hultin, 1984). As a result, the protons that are produced during glycolysis and during hydrolysis of ATP to ADP cause a significant (pH 7.2 to pH 5.5) decrease in the intracellular pH (Honikel & Hamm, 1974). Not only the extent of post-mortem metabolism and its associated decrease in pH are important factors that govern meat palatability. Since protein denaturation is a function of both pH and temperature, also the rates at which these processes take place are of relevance (Ashgar & Pearson, 1980). In a recent contribution (Vogel et al., 1985) we have introduced phosphorus-31 N M R as a convenient monitor for studying simultaneously the extent and the rate of the post-mortem metabolism of ATP, CP, sugarphosphates (SP) and inorganic phosphate (Pi) in bovine muscles. Noninvasive N M R is presently widely accepted as a useful tool for the study of metabolism in living cells, excised organs and intact animals (for reviews see Ingwall, 1982; Meyer et al., 1982, Gadian, 1983, Lundberg & Vogel, 1987). Although the N M R spectra themselves are semiquantitative, the outcome of the experiments can be converted to quantitative numbers by using conversion factors (Vogel et al., 1987). The results reported in our earlier account demonstrated an excellent qualitative and quantitative agreement between the course of post-mortem metabolism as determined by N M R and by classical enzymatic methods (Vogel et al., 1985). The intracellular pH is an additional parameter that can be deduced directly from the 31p N M R spectra. Thus, lactate production and glycogen breakdown are the only relevant parameters in post-mortem metabolism that cannot be determined directly using 3IP_NM R studies. In an accompanying paper we have demonstrated, however, that these entities can be measured conveniently by natural abundance carbon-13 or proton N M R studies (Lundberg et al., 1986). For part of this contribution we have used 3~p-NMR to study the postmortem metabolism in fresh samples obtained from porcine and ovine species, where the post-mortem rates of metabolism and the associated rate

31p NMR of post-mortem metabolism

3

of the pH decline are expected to be higher than with bovine muscles. The second part of this study concerns post-mortem metabolism during thaw shortening in thawing samples of prerigor bovine muscle. This so-called thaw shortening is caused by a supercontraction which takes place during thaw of most muscles that have been frozen in a prerigor state. Although these phenomena are well documented, little is known (at present) about the details of the post-mortem metabolism that takes place during thaw shortening, partly because it happens so rapidly. Here we show that it can be followed conveniently by 31p NMR.

EXPERIMENTAL PROCEDURES

Samples Dark and light thigh muscles (Musculus adductor and M. semimbranosus, respectively) from lamb and pig carcasses were taken on the slaughter-line and placed in tubes of acrylic plastic (~b 20 mm) and of glass (~b 10 mm). The pig samples were placed in tubes about 40min after slaughter. Then the samples were transported by car from the slaughterhouse and placed into the probe in the NMR-machine at about 1-1 h after slaughter. The samples were transported in an insulated box at a temperature ranging from 8° to 15°C. The lambs were slaughtered in the slaughterhouse individually by hand. These samples arrived at the laboratory about 1.0 h after slaughter. Pieces of muscles from cow were rapidly frozen in liquid nitrogen as fast as possible after slaughter ( < 15 min). The samples were frozen in tubes of acrylic plastic (~b 20 mm) as described above, and stored at - 80°C until the N M R experiments were performed. Before each N M R experiment the tube with sample was covered with a small piece of plastic film (parafilm).

NMR spectroscopy 3~p-NMR spectra were recorded on a homebuilt 6 Tesla spectrometer (described by Drakenberg et al., 1983) operating in the Fourier Transform mode with quadrature detection. During the experiments two different homebuilt horizontal, solenoidal probes were used (q5 10 mm and q520 mm, described respectively in Vogel et al., 1983 and Vogel et al., 1987). The irradiation frequency was 103-2 MHz and the sweep width was 20000Hz. Radio frequency pulses of 50 ps (90 ° and 50 °, respectively) were used and the recycling time was 8 s (sufficiently long to allow for complete T~ relaxation). Normally, 160 scans were collected for each spectrum which gave a total time (including the time needed to change the samples) of

4

Peter Lundberg, Hans J. Vogel, Stefan Fabiansson, Hdkan Ruderus

25 min per spectrum. 8 kbyte memory was used to accumulate the free induction decay. All spectra were plotted with an extra line broadening of about 30Hz to decrease noise. All peaks are referenced to 85% H3PO, L (0 ppm) and integrated by cutting and weighing. For the post-mortem metabolism of fresh meat, both 10 mm and 20 mm probes were used. The experiments were performed at 16°C and 25~C. The lower temperature was maintained by passing cooled nitrogen gas through the isolated probe. For the experiments with thawing meat only the 20 mm probe was used at 25°C external temperature. For the latter experiments sample sizes ranged from 5 to 7 g. The temperature during thawing was measured in separate experiments with a thermoelement placed in a tiny borehole in the sample, which was placed in the normal fashion in the N M R magnet. pH measurements

Intracellular pH measurements were performed as described earlier (Moon & Richards, 1973; Vogel & Brodelius, 1984; Vogel et al., 1985, 1987). The method relies on the use of the chemical shifts of glucose-6-phosphate (G6P) and Pi as a sensitive monitor of pH. The pK~ of G6P is around 6-2 making this resonance a good pH indicator between 6-8 and 5.6. In this study we have measured pH values below 6-8 and thus we have relied on using the chemical shift of G6P resonance as the more accurate pH indicator. RESULTS Post-mortem metabolism in fresh meat

Figure 1 depicts the 31p-NMR spectra of meat samples obtained from three different species. The same six resonances can be discerned in all three instances. The assignment of the resonances is (from left to right) sugar phosphates (SP), inorganic phosphate (Pi), creatine-phosphate (CP) and the ATPT, ~ and fl phosphorus resonances. The pyrophospho group from NAD(H) contributes a small amount of intensity to the resonance of ATP~. The chemical shift of the ATPfl resonance is similar in all three cases, thus suggesting that the Mg 2+-saturation of ATP is more than 90% and that it is very similar in all three cases (for discussion see Hellstrand & Vogel, 1985). Any free intracellular ADP would contribute to the intensity of the ATP:~ and 7 resonances. (Protein-bound ADP would give rise to a very broad resonance, that probably would escape detection here.) However, the ATPfl and ATP7 resonances were of similar intensity at every point during

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Fig. 1. 31p-NMR spectra of cow (A), lamb (B) and pig muscle (C) at 1.2h after slaughter. The assignment of the resonances is discussed in the text and is indicated in panel A. The spectra for panels B and C are obtained with the 20ram probe and that of panel A with the 10mm probe.

the post-mortem metabolism, indicating that the amount of free ADP is always less than the error of the determination (<5%). The main component of the unresolved sugar phosphate resonance is gtucose-6phosphate at the beginning of the post-mortem metabolism in bovine muscles (Vogel et al., 1985). At the end of post-mortem metabolism it may contain a considerable amount of inosine monophosphate (Fabiansson & Laser-Reutersw/ird, 1985), which, as a phosphomono-ester, would have a chemical shift in this region. Although the spectra for all three species are taken at approximately the same time (1.2h) after slaughter, there is a marked difference in the intensities of the resonances. For example, the CP resonance is the most intense in spectrum A, but it is barely visible in spectrum C. Moreover, the SP and Pi resonances in spectra B and C have shifted upfield as compared

6

Peter Lundberg, Hans J. Vogel, Stefan Fabiansson, Hhkan Ruderus

with spectrum A, indicating a significantly lower pH in the pig and lamb muscles than in the cow muscles. Thus simple inspection of these three spectra already reveals that the rate of post-mortem metabolism is in the following order: pig muscle > lamb muscle > cow muscle. A detailed course of the post-mortem metabolism is shown in Figs 2 and 3 for pig and lamb, respectively. In order to avoid complications in calibration introduced by the different sample and probe sizes and the varying efficiencies in probe tuning, etc., we have expressed the N M R resonances as a percentage of the total amount of N M R visible phosphorus. This value did not change over a 10-h period for pig or lamb samples (data not shown). A figure corresponding to Figs 2 and 3, but for bovine muscle, has been reported earlier (Vogel et al., 1985). In all three cases the CP is the first resonance to 100 f,= o

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Fig. 2. Time course of the post-mortem metabolism of pig muscle as determined by 3~p. NMR. Pi ([Z), ATP (A), sugar phosphates (O) and intracellular pH (A). The data points are the average of three separate experiments. CP had been depleted almost entirely before we started our data collection and is not plotted here.

disappear. Once the CP pool has been depleted, the levels of ATP start decreasing. The sugar phosphate level remains fairly constant over the whole period and the Pi level increases as a result of the hydrolysis of phosphorylgroups from the CP and ATP. The intracellular pH decreases from 6.1 to 5-5 for the pig muscles, during the time course in which we were able to measure. For lamb muscles we registered a decrease from pH 6.4 to 5.6. The corresponding values for the bovine muscles were pH 6-9 and 5"6 (Vogel et aL, 1985). The levels of the phosphorylated metabolites remained constant after about 3 h for the pig muscle, after 5 h for the lamb muscle and alter 12 h tbr bovine muscles. When the post-mortem metabolism was followed at 16:C, rather than 25"C, the corresponding rates were slower by slightly less than a factor of 2 for pig and lamb samples. A comparison

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Fig. 3. Time course of the post-mortem metabolism of lamb muscles as determined by 3'p-NMR. Pi (T]), ATP (A), sugar phosphates (©) and intracellular pH (A). The data points are the average of three separate experiments. CP had been depleted almost entirely before we started our data collection and is not plotted here.

between ground and untreated lamb meat showed an increase of at least fivefold for the rate of the post-mortem metabolism in ground meat (data not shown). In all these instances the post-mortem metabolism followed the same course and no other metabolites were detected. Bendall (1979) reported that he observed a linear relationship between the ATP level and the pH or lactate level independent of the rate of glycolysis. Figure 4 shows that if we plot the N M R determined content of I

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8

Peter Lundberg, Hans J. Vogel, Stefan Fabiansson, Hhkan Ruderus

ATP (as a percentage of total phosphorus) versus the intracellular pH as determined by N M R (using the pH data obtained with the glucose-6phosphate standard curve) for more than forty individual spectra we also find a straight line. A similar plot using the data obtained earlier for bovine muscle (Vogel et al., 1985) showed the same trend, but gave a much poorer correlation (data not shown). Post-mortem metabolism upon thawing of frozen prerigor meat In frozen meat the rotational mobility of metabolites should be restricted because of the presence of the ice-lattice. Consequently, resonances for the phosphorylated metabolites are expected to be broadened to the extent that they cannot be observed. This is indeed the case. Figure 5A shows that at - 2 0 ° C only two relatively broad resonances are observed, one for CP and one is a composite peak for sugar phosphates and Pi. Upon thawing to - 5 ° C , spectra such as in Fig. 5B were obtained, where obviously the line width of the resonances (determined at half height) has decreased considerably. The total intensity of the resonances has increased and three new resonances for ATP have appeared. At 0°C these changes continue (see Fig. 5C). At that point the post-mortem metabolism appears to have started since the Pi resonance is increasing while the CP resonance is decreasing.

10 0 -,0 -2° pp° Fig. 5. ~IP-NMR spectra obtained for the same sample of frozen bovine meat upon thawing. (A) - 20°C, IB) - 5C, (C) 0" to -1-1~C, (D) 15~C.The assignment of the resonances is indicated in the Figure.

31p N M R of post-mortem metabolism

9

When the center of the sample has reached a temperature of about 15°C (this is at approximately 1.2 h after the frozen sample is placed into the N M R probe which is at 25°C), post-mortem metabolism has obviously been completed because CP and ATP are no longer present and the Pi resonance has moved to a chemical shift indicative of a very low pH value. Figure 6a shows the temperature as measured with the help of a thermoelement at the center of a meat sample which was otherwise treated exactly the same as samples that were analyzed by NMR. Note, in particular, the large plateau region around 0°C. Figure 6b shows the line width (measured at half height and not corrected for the extra introduced broadening) of the Pi resonance during a typical thawing experiment. Obviously, the line width reaches a plateau as soon as the sample reaches 0°C. It is thus likely that the broadening is the result of the restrictions in motion that are introduced by the ice crystal formation which takes place at lower temperatures. Because of anticipated differences between samples with and without ice we checked and adjusted the probe-tuning before each individual spectrum. Large adjustments were generally needed. Since we were worried that faulty probe-tuning could give rise to reduced spectral intensities, we performed the control experiment depicted in Fig. 6c. For 10

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10

Peter Lundberg, Hans J. Vogel, Stefan Fabiansson, H~kan Ruderus

this experiment we used three different samples of frozen postrigor meat in which there is only a Pi resonance (pH 5.5) detectable. All metabolic activity has stopped in such meat and the intensity of the Pi peak is not expected to change, unless the difference in sample inductivity causes changes in the sensitivity of the N M R experiment. Fortunately, the data presented in Fig. 6c show that there is very little variation in this intensity as long as the N M R probe is retuned between all individual experiments. Figure 7 shows in detail the post-mortem changes in the levels of the metabolites CP, ATP and Pi as well as the changes in pH. Very few changes are observed before the sample reaches 0°C. This was further confirmed by placing similar frozen samples in the N M R probe which was cooled to temperatures of - 5 ° C or lower. No changes could be detected in the 3tp. N M R spectrum within a time course of 2 h in these samples. Post-mortem metabolism could, however, be detected in a sample placed at 0:C. The course of the post-mortem metabolism, as depicted in Fig. 7, is very similar to those discussed earlier. The CP level decreases rather rapidly. The ATP level remains constant until CP is depleted. Pi increases as a result of the hydrolysis of these metabolites. While these processes take place, the intracellular pH, as measured from the chemical shift from Pi, drops from 7.2 to 5"5. The largest pH drop occurs after the sample has warmed up to over 0°C. Thus, the course of the post-mortem metabolism in thawing meat that has been frozen in the prerigor state is similar to that of fresh bovine 100

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Fig. 7. Time course of the post-mortem metabolism of frozen cow muscle during thawing. The probe was at 25~C. The amounts ofCP (C)), ATP (V) and Pi + SP ( I ) are indicated, as well as the intracellular pH (A). To simplify the comparison the temperature curve from Fig. 6a is also included in the Figure. The points in this Figure represent the average of five different independent experiments. Because of the way in which the experimental points are calculated as a % of total phosphorus, it appears as if Pi + SP and CP decrease initially within the first 15 min, while they remain constant in fact. Similarly, the intracellular pH is underestimated in the first point because the Pi + SP peaks are not separated at this point.

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meat. However, once the sample reaches 0°C all the changes take place in less than 1 h (see Fig. 7) which is at least a factor of 10 times faster than is observed normally with beef muscles (Vogel et al., 1985). It is likely that this extremely fast post-mortem metabolism fuels the supercontraction which is the cause of the thaw shortening. Indeed, all the frozen meat samples (except those used for Fig. 6c) reduced to about 60% of their original length in the course of the thawing experiment. Figure 8 shows the total amount of phosphorus (in arbitrary units) in the course of a typical thaw experiment. Note that this total amount first increases as the meat becomes unfrozen, which is followed by a gradual 30% decrease in the total amount. DISCUSSION Our earlier studies of post-mortem metabolism in flesh bovine samples had already demonstrated that 31p-NMR is a useful tool to follow postmortem changes in muscles from slaughter animals (Vogel et al,, 1985). The studies presented here confirm and extend this notion. Five metabolic variables determine, to a large extent, the anaerobic metabolism in muscle tissues. These are: the levels of ATP, phosphocreatine, glycogen, lactate and the intracellular pH. Three of these parameters can be obtained directly from 3tp-NMR spectra. In addition, the lactate content can, in principle, be deduced, since the intracellular pH is linearly related to the amount of lactate production (Bendall, 1979; Fabiansson & Laser-Reutersw~ird, 1985; Lundberg et ak, 1986). Furthermore, although the glycogen is important as an energy source, it does not play a regulatory role since the amount in most muscle tissues is such that it will not be depleted by the post-mortem

Peter Lundberg, Hans J. Vogel Stefan Fabiansson, Hfkan Ruderus

Letabolism in most animals (Ashgar & Pearson, t980; Fabiansson & aser-Reutersw~ird, 1985; Lundberg et al., 1986), although it may happen in le case of the pig (Hultin, 1984). Rather, the drop in the intracellular pH om 7"0 to 5-5 will reduce the activity of glycogen phosphorylase and other ycolytic enzymes. Thus, the pH decrease, rather than the depletion of ycogen, is the regulatory mechanism that blocks further post-mortem tetabolism. As a consequence, meat can be chilled or frozen without ~ving to worry about the possibilities of thaw-shortening when all the TP has disappeared and the intracellular pH has fallen to a reasonably ,w level. The linear correlation observed here between pH and ATP ~ntent (Fig. 4) indicates that, in fact, it is not even necessary to perform an TP measurement but that a simple pH measurement would be sufficient decide on the optimal time to cool down the carcass. Because of the unavoidable time lag between slaughter and the moment : which the sample can be analyzed by N M R (> 1 h) we have only been Me to follow the last part of the post-mortem metabolism in lamb and pig Luscles. This is not an inherent problem of N M R sensitivity since the time :solution of the N M R experiment is sufficient to follow the extremely fast 9st-mortem metabolism in frozen prerigor muscle (see Fig. 7), but it is tther a problem of logistics with the N M R spectrometer being far :moved from the slaughterhouse. Although one could consider studying ssue extracts that are prepared from fresh samples during this time delay, : doing so the information about the intracellular pH would be lost which ould make this analysis a lot less useful. Only a few N M R studies of living tissues that were frozen have been ~ported (Chance et al., 1978, Storey et al., 1984). Nevertheless, very similar ~enomena to those reported here have been observed where the line width r resonances decreases dramatically with increasing temperatures. Thus it reins likely that the presence of the ice lattice significantly reduces the obitity of the metabolites in frozen tissues. As a result, some residues give se to broadened N M R peaks (CP and Pi + SP), whereas others are so :oad that they escape detection (ATP). The latter compounds reappear in te spectra as the temperature approaches 0:C. The data presented in Fig. 8 quire some further discussion, however; whereas the initial increase in ,tal phosphorus during the first half hour can be rationalized in terms of a •eater degree of motional freedom for metabolites in the absence of ice ystals, it is more difficult to explain the subsequent 30% decrease in the ,tal amount of phosphorus-containing metabolites. The experiment :picted in Fig. 6c makes it unlikely that this is completely caused by a ulty adjustment of the NMR-probe tuning. It can also not be excluded at the reduction in muscle size caused by the supercontraction is related this observation, but one would predict that this would lead to an

3tp NMR of post-mortem metabolism

13

increase, rather than a decrease. Since the onset of the decrease coincides with the point where the temperature reaches 0°C, it could be of metabolic origin. Inspection of the absolute intensity plots (data not shown) of the experiment depicted in Fig. 7 shows that this decrease in intensity mainly originates in a decrease of the Pi resonance. Thus, the most likely explanation is that some of the Pi is rendered N M R invisible (too broad to be detected), possibly through precipitation with metal ions, through binding to large macromolecules, or through sequestration into viscous organelles. It has been reported that the total amount of ADP, as determined in bovine muscle extracts, remains at 20% of the initial level of ATP during the first 10h of the post-mortem metabolism (Fabiansson & LaserReutersw~ird, 1985). Therefore, it is noteworthy that we do not detect any amount of free ADP in our N M R spectra. Thus, we have to conclude that ADP remains in a bound state, probably as part of the myofibril, during post-mortem metabolism. In conclusion, the major outcome of these studies can be summarized as follows, The post-mortem metabolism, as studied by 3tp-NMR in fresh skeletal muscle tissues obtained from different species, always follows the same course. Interestingly, this also applies to the post-mortem metabolism which takes place upon thawing of meat that was frozen in the prerigor state. The final pH is usually around 5.6. Furthermore, in all instances the steady-state levels of ATP are kept relatively constant, until the CP level has dropped below that of ATP. This mode of action is in support of a simple model where CP functions as an energy reservoir for ATP. Other unexpected metabolites (such as glycerol-3-phosphoryl-choline and serineethanolamine-phospho-diester) have been shown by 3tP-NMR studies to be present in considerable amounts in certain tissues and cells, including skeletal muscle (Glonek et al., 1980). In none of the muscles that we have studied did we encounter any of these compounds. Notwithstanding these similarities, the rates of post-mortem metabolism are significantly different for samples obtained from different species that have been treated in the same way after slaughter. The rates that we measured can be ordered in the following manner: thawing of frozen prerigor muscle > porcine muscle > ovine muscle > bovine muscle. Incubation of fresh samples at lower temperatures (16°C) than used throughout (25°C) gives a reduction in these rates. REFERENCES Ashgar, A. & Pearson, A. M. (1980). Adv. Food Res., 26, 53-165. Bendall, J. R. (1979). Meat Science, 3, 143.

Peter Lundberg. Hans J. Vogel Stefan Fabiansson, 'Hdkan Ruderus

B., Nakase, Y., Bond, M., Leigh, J. S. & McDonald, G. (1978). Proc. L Acad. Sci. USA, 75, 4925-34. ~erg, T., Forsen, S. & Lilja, H. (1983). J. Magn. Reson., 53, 412-20. ;on, S. & Laser-Reutersw~ird, A. (1985)• Meat Science, 12, 205-15. D. G. (1983). Annu. Rev. Biophys. Bioeng., 12, 69-89. T., Burr, C. T. & Barany, M. (1980). In: N M R m medicine (G. Damadian )), Springer Verlag, Berlin, 121-59. ad, P. & Vogel, H. J. (1985). Am. J. Ph.vsioL, 248, C320-C329. K. & Hamm, R. (1974). Fleischwirtschaft, 54, 557. q. O. (1984). J. Chem. Educ., 61,289-98. J. S. (1982). Am. J. Physiol., 242, H729-H744. '~ P. & Vogel, H. J. (1986). Anal. Biochem. (In press.) g, P., Vogel, H. J. & Ruderus, H. (1987). 34eat Science, 18, 133-60. R. A., Kushmerick, M. J. & Brown, T. R. (1982). Am. J. PhysioL, 242, C1 I. ~.. B. & Richards, J. H. (1973). J. Biol. Chem., 284. 7276-78. 7,. B., Miceli, M., Butler, K. W., Smith, I. C. P. & Deslauriers, R. (1984). Eur. 'iochem., 142, 591-5. F. J. & Brodelius, P. (1984). J. Biotechn.. !. 159-70. [. J., Lilja, H. & Hellstrand, P. (1983). Biosci. Rep.. 3. 863-70. • J., Lundberg, P., Fabiansson, S., Ruderus, H. & Tornberg, E. (1985). Meat ~zce, 13, 1-17. I. J., Brodelius, P., Lilja, H. & Lohmeier-Vogel, E. M. (1987). Methods vmoL, 135 (In press).