Effect of Vitamin E on changes in Phosphorus Compounds assessed by P NMR Spectroscopy and ATPase from postmortem Muscle samples and Meat quality of Pigs

The effect of addition ofvitamin E (a-tocopherol) to pig diets on muscle metabolism of phosphorus Compounds, ATPase activity and meat quality was studied. Experimental pigs were fed with a diet supplemented with vitamin E (200 mg a-tocopherol/kg diet) for 60 days before slaughtered (110 kg live weight). Longissimus muscle (LD) Vitamin E level was more than twice higher (P<0.01) in pigs supplemented with vitamin E Changes of muscle phosphorus Compounds like sugar phosphate (SP), inorganic phosphate (Pi), phosphoereatine (PCr), and adenosinetriphosphate (ATP) assessed by P NMR spectroscopy (15 min post mortem) differed between control pigs and vitamin E administered pigs. Significantly lower (PO.05) values of SP and significantly higher values of PCr were found in pigs administered with vitamin E. Efficiency of muscle energetic metabolism measured as index PCr/Pi was higher in pigs supplemented with vitamin E. ATPase activity of longissimus muscle (LD) was not influenced by vitamin E in diet. Drip loss of LD measured 24 h post mortem and conduetivity of semimembranosus (SM) and LD measured 3 h post mortem (PO.05) improved by administration with vitamin E. Differences between pH of LD and SM muscles measured 45 min post mortem were not significantly influenced.


Introduction
The beneficial effect of dietary supplementation of vitamin E on some aspects of meat quality has been reported by various investigators.The stability of lipid and colour in beef (ARNOLD et al., 1993), pork (MONAHAN et al., 1992), and a reduction of drip loss from pork chops following frozen storage (ASGHAR et al., 1991) could be achieved by dietary supplementation of vitamin E. Oxidative processes may contribute to the loss of membrane integrity.This oxidation leads to a decrease in fluidity and disruption of normal membrane structure and function, and may affect the ability of the membrane to act as a semi-permeable barrier (STANLEY, 1991).Positive effects vitamin E on the oxidative stability and quality of pig meat (drip loss) were reported by BUCKLEY et al. (1995) and on the amount of fluid in muscles by LAURIDSEN et al. (1999).Some investigators did not find significant differences between drip loss of vitamin E supplemented and control porcine muscles (JENSEN et al., 1997;DUFEY, 1998;HONIKEL et al., 1998), whereas the effect of vitamin E supplementation observed by CHEAH et al. (1995b) depended on the muscle investigated.Also, the effect of vitamin E supplementation on drip loss of bovine muscles seems to depend on the muscle studied (DEN HERTOG-MEISCHKE et al., 1997).As was shown (MITSUMOTO et al., 1998) appropriate feeding, dose of vitamin E, and concentrations in muscles should be achieved to be effective in reducing drip losses from fresh beef Steaks.There were proposed more potentially modifying factors influencing muscle metabolism, malignant hyperthermia (MH) and meat quality in pigs as was discussed by FLETCHER et al., (1993).Abnormality in the antioxidant defence system could be one ofthe factors (DUTHIE et al., 1991).It was shown that the capacity of muscle energetic metabolism could also be an important factor influencing and modulating the MH Syndrome and meat quality (LAHUCKY et al., 1993;KOHN et al" 1998).Heterozygotes can form a metabolically distinet phenotype between homozygotes negative and homozygotes MH susceptible pigs as was shown using phosphorus nuclear magnetic resonance ( 3I P NMR) spectroscopy (MOESGARD et al., 1994;LAHUCKY et al., 1998).Phosphorus NMR spectroscopy has previously been applied in studies on skeletal muscle energetic metabolism using frozen or fresh bioptates (SHEN et al., 1992;LAHUCKY et al., 1993).The objective of this study was to determine the effects of dietary vitamin E supplementation on changes in phosphorus Compounds assessed by NMR spectroscopy and also overall ATPases as major energy consumers from postmortem muscle samples.Meat quality values of pigs were evaluated as well.

Animals and sample preparations
In total 18 pigs were used in this experiment.They originated from lines of Large White and crossbred Large White x Pietrain pigs.The RYR-1 genotype (FUJII et al., 1991) of this animals was determined by a DNA based test described previously (LAHUCKY et al., 1998).Two groups (experimental, control) with 5 normal and 4 heterozygotes on MH Syndrome in each group were taken in this experiment.The pigs (3 barrows and 6 gilts in each group) were penned in double boxes at institute facilities to minimise the influence of stress.At the beginning of vitamin E administration (70 ± 2 kg live weight) water holding capacity (WHC) and pH from bioptate (longissimus muscle) were estimated (CHEAH et al., 1993) from heterozygote pigs.Values of WHC and pH did not differ significantly (PO.05) between heterozygotes of control and experimental group.Experimental animals were supplemented daily with 200 mg vitamin E (Slovakofarma, Hlohovec) per kg of Standard diet for 60 days before slaughter.The animals were electrically stunned and slaughtered at 110 kg in the slaughter house ofthe institute in Nitra (transportation about 200 m).Immediately after the exsanguination (10 min) a sample from the longissimus muscle was removed (referred to as 0 h sample).At 24 h post mortem the next sample was taken after chilling.All these samples were frozen in liquid nitrogen, stored at -70° C, homogenised and treated as described by KÜCHENMEISTER et al. (1999).From the right side of longissimus muscle (last rib) 15 minutes after slaughter a sample of approx. 1 g using was taken out by a biopsy instrument (Biotech, Nitra), immediately frozen and stored in liquid nitrogen until analysed.The pH value of the carcass (longissimus -between 13th and 14th rib, semimembranosus muscle-middle) was determined at 45 min post mortem using combined pH electrode (Ingold).Electrical conduetivity was measured with a Teepro Quality instrument (Germany) at 3 h and 24 h post mortem.Drip loss analysis was made aecording to HONIKEL (1998) 24 h post mortem.The experiments were in accordance with the institutional guidelines for animal care (Research Institute of Animal Production, Nitra, 1998).

a-Tocopherol determination
A samples of longissimus muscle (frozen in liquid nitrogen and stored for five months at -70° C) were analysed in duplicate for a-tocopherol content.

Vitamin E
The concentration of vitamin E of blood and muscle were measured by HPLC (BERLIN et al., 1994).A mixture of 1.5 ml muscle homogenate or 3 ml plasma, 2 ml absolute ethanol and 0.5 ml 10 % ascorbic acid was heated to 70° C for 5 minutes.After adding 1 ml 10 n KOH, the mixture was ineubated at 70° C for 30 minutes.After cooling, 5 ml n-hexane was added for extraction.The solvent was removed by evaporation under nitrogen, and the vitamin E was immediately resolved in absolute ethanol and assayed by HPLC.HPLC analysis was performed with the mobile phase methanol with a flow rate of 1 ml/min and a Lichrospher RP 18 column with precolumn (Müder & Wocherle Chromatographietechnik Berlin, 12.5 x 0.4 cm, 5 mm).Detection was performed by fluorescence at 292 nm excitation/336 nm emission.Peaks were quantified upon calibration with authentic samples of a-tocopherol (Sigma, Deisenhofen).

ATPase activity measurements
The total ATPase activity of the muscle homogenates was measured spectrophotometrically with a coupled enzyme assay at 30 C by a modified method described previously (SIMONIDES et al, 1990), but without the inclusion of any Inhibitors.The reaction mixture consisted of 20 mM HEPES (pH 7.5), 100 mM KCl 2 mM ATP, 5 mM MgCl 2 , 1 mM EGTA, 1 mM CaCl 2 , 0.2 mM NADH, 16 IU lactate dehydrogenase, 10 IU pyruvate dehydrogenase, 10 mM phosphoenolpyruvate and 2 M Ca lonophore A23187.The free Ca 2+ concentration was calculated to be about 10 M. After a preincubation for 10 min to equilibrate the temperature, 10 1 homogenate were added and the decrease ofthe absorbance followed for about 5 min.

Phosphorus nuclear magnetic resonance spectroscopy
The biopsy sample (approx. 1 g) was introduced into a 10 mm diameter tube (maintained at 39°C) filled with deuterated water (D 2 0) for NMR measurements The P NMR^pectrum was recorded at 121 MHz on a VXR 300 (Varian) spectrometer.The total P spectrum was recorded with a sweep width of 3932.4Hz and 45 pulses of 35.0 s The recycle time was 0.8 s.Each spectrum was a result of 512 transients.An exponential line broadening of 20 Hz was used as internal reference at -2.47 ppm.The time of aecumulation per spectrum was 7.6 min.Out of a number of spectra for calculation only the first and second were used.The levels of the individual phosphorus Compounds were expressed in percentage of the total content of phosphorus Compounds as described previously (LAHUCKY et al.,

Statistical analyses
We used a three-way cross Classification model with three factors: feeding group (two levels: with vitamin E and without) Status (two levels : homozygotes negative and heterozygotes) sex (two levels: barrows and gilts) and interaction effects feeding group x Status and feeding group x sex.For the influence ofthe sampling time a repeated measurement model was used.The analysis was done by means of Statistical Software package SAS©, procedure GLM.

Results
Figure 1 shows series of 3I P NMR spectra obtained from longissimus muscle of control (a) and of vitamin E (b) administered pigs.The spectra of pig muscle contained six peaks corresponding mainly to resonances of sugar phosphate (SP), inorganic phosphate (Pj), phosphocreatine (PCr), and the three phosphate groups of adenosinetriphosphate (ATP), respectively.The signals of PCr and ATP decreased in the time course, whereas Pj and SP increased to a lesser extent.The rate of changes in concentration of phosphorylated Compounds varied between pigs.In the vitamin E supplemented and in the control pigs there was still a high ATP level at the beginning ofthe NMR measurements (1.spectrum) and remained high or decreased tili the end of measurements (3.spectrum).By contrast, the PCr level was lower from the beginning and was almost exhausted at the end of measurements, .-*/VAJVWV, i ' ' ' ' ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' P ' ' ' I ' ' ' ' I  mainly in samples taken from control pigs (Fig. la).That's why we have calculated only the results from 1. and 2. spectrum.The level of SP and Pj were higher at beginning of measurements and SP was increased in a higher extent in control pigs tili the end of measurements (3.spectrum).
As follows from the results introduced in Table 1 significant differences (PO.05) of SP were received at the 1. and 2. Spectrum.Differences in Pj of 2. spectrum and also differences of PCr values at 1. spectrum (PO.05) and second spectrum(PO.Ol) were significant between control and vitamin E supplemented group.Values of ATP were also lower in longissimus samples of control pigs but differences were not significant (P>0.05).
Figure 2 illustrates the change of the ATPase activity of longissimus muscle determined just after slaughter (0 h) and 24 h post mortem from control and vitamin E supplemented pigs.The ATPase activity did not differ between experimental groups immediately after slaughter and 24 h post mortem as well.There was no significant decrease of activity from the 0 h sample to the 24 h samples from vitamin pigs whereas this decrease was significant in samples from control pigs (Fig. 2).The content of vitamin E (a-tocopherol) of the longissimus muscles was determined and the results are presented in Table 2. Dietary vitamin E level in pigs supplemented with vitamin E was more than twice higher if compare to control pigs (PO.01).Meat quality values measured as pH, electrical conduetivity and drip loss of longissimus muscle and pH and electrical conduetivity of semimebranosus muscle (Table 2) showed differences between control and with vitamin E administered pigs.
There was a tendency (P=0.1) of lowering the drip loss in longissimus muscle of pigs with supplementation of vitamin E. Dietary vitamin E levels significantly (PO.05) affected electrical conduetivity of longissimus and semimebranosus muscle measured 3 h post mortem.Differences of pH measured 45 min post mortem oilongissimus and semimebranosus muscles were not significant (P>0.1).

Discussion
The higher level (PO.01) of a-tocopherol in longissimus muscle of vitamin E supplemented pigs (200 mg/kg diet for 60 days) is in agreement with those of other authors (BUCKLEY et al., 1995;O'SULLIVAN et al., 1997).Tissue levels of cttocopherol were 2.5 to 3.0 times higher in pigs fed the supplemented diet (200 mg/kg diet for 125 days) than in those on the basal diet (30 mg/kg diet) (BUCKLEY et al., 1995).Similar results were reported by ASGHAR et al. (1991) for pigs reeeiving 10, 100, or 200 IU vitamin E per kg of feed.We have no information about the basal level of a-tocopherol in the diet.This and/or longer storage ofthe samples (five months, at -70° C, non vaeuumed, ones thawed and frozen) could be reasons for the overall lower values of a-tocopherol in longissimus muscle from both supplemented and control pigs obtained in our experiment.It is known that a-tocopherol levels in porcine tissues depend on the supplementation time.When pigs were given a diet supplemented with 200 mg a-tocopheryl acetate per kilogram of feed, a-tocopherol levels increased with supplementation time up to 91 days in tissues (BUCKLEY et al., 1995).It is believed that incorporating vitamin E into subcellular membranes increases the antioxidant capacity ofthe system and possibly also increases their physical stability (BUCKLEY et al., 1995).Different changes in breakdown of PCr and ATP were measured comparing control and vitamin E supplemented pigs (Fig. 1, Tab. 1).In every case PCr and ATP decreased in the time course, whereas SP increased.Changes of Pj were different, the level of this Compound reaching a plateau at 1. spectrum (muscle sample taken 15 min post mortem and 12 min NMR spectrum).That could be consistent with results introduced earlier (LAHUCKY et al., 1993).After 20-30 min ineubation usually a plateau of Pj level was reached.Breakdown of PCr was higher in the control group with significant differences in both spactra: 1. spectrum (PO.05) and 2. spectrum (PO.01).The level of SP was significant lower (PO.05) in samples from vitamin E group (mainly in 1. spectrum).The ratio of free inorganic phosphate to phosphocreatine (Pj/PCr) was proposed as an indicator ofthe energy State of muscle tissue in various conditions and the inverse ratio (PCr/Pj) was also used (LAHUCKY et al., 1993).Rate of breakdown of PCr seems to be higher in pigs supplemented with vitamin E as follows from the level of PCr in control vs. vitamin E group (Tab. 1) and/or higher ratio PCr/Pi (1.spectrum -0.075 vs. 0.013) in the vitamin E group.Higher variability was found in ATP and differences between groups were not significant (P>0.05).This was also supported by the results of the ATPase activity measured from longissimus muscle just (0 h) and 24 h after slaughter (Fig. 2).ATPase activity from pigs supplemented with vitamin E were higher at beginning (0 h) and 24 h post mortem but differences between groups were not significant (P>0.05).However, the decrease of ATPase activity from control pigs from 0 h to 24 h post mortem was significant, but not so in vitamin E supplemented samples (Fig. 2).As possible mechanism for the beneficial effect of vitamin E supplementation on drip loss has been suggested that a-tocopherol could preserve integrity of the muscle cell membrane by preventing oxidation of membrane phospholipids during refrigerated storage (ASGHAR et al., 1991).This could be consistent with higher efficiency of energetic Compounds in muscle (lower breakdown of PCr, higher level of index PCr/Pi) and lowering of conduetivity (3 h post mortem) in animals administered with vitamin E. The effect of vitamin E supplementation on drip loss seems to depend on the muscle studied (DEN HERTOG-MEISCHKE et al., 1997).Their results suggested that vitamin E supplementation may have both positive and negative effects on drip loss of meat, depending on muscle studied.Results received (Tab.2) on pH, conduetivity, and drip loss supported positive effects of vitamin E. Differences in conduetivity of longissimus and semimebranosus muscles measured 3h post mortem between control and the vitamin E administered group were significant (PO.05).There was a tendency in improving drip loss of longissimus muscle (P=0.1).Differences in pH of LD and SM muscles measured 45 min post mortem were not significant (P>0.1) between groups.Using a higher level of vitamin E supplementation (500 mg/kg diet) administered for 46 days could reduce drip loss in unfrozen longissimus thoracis in heterozygotes and in normal on malignant hyperthermia pigs as was shown by CHEAH et al. (1995).Authors indicated that part of vitamin E effectiveness in improving the WHC of longissimus thoracis and in preventing changes in meat quality is due to inhibition of phospholipase A 2 activity connected with hydrolysis of phospholipids and stability of mitochondrial membranes.This could be also supported by our results of higher efficiency muscle energetic metabolism of pigs administered higher level of a-tocopherol.However, there are contradictionary results about posi-tive effects of vitamin E supplementation on drip loss (BUCKLEY et al., 1995), or the amount of fluid in muscles (LAURIDSEN et al., 1999) and some investigators were not able to find significant differences between drip loss of supplemented and control porcine muscle (JENSEN et al., 1997;HONIKEL et al., 1998).MONAHAN et al. (1994) have shown that membrane lipid oxidation and drip loss of pork are not directly related.In agreement with HONIKEL et al. (1998) membrane changes (oxidative changes) post mortem have not to be in relation to some quality values (e.g.drip loss, conduetivity) when muscle is stored for a longer time.The influence of vitamin E supplementation on conduetivity and drip loss of muscles was not always consistent in our experiment.It is believed that conduetivity and drip loss could be affected not only by dietary vitamin E supplementation but also by other factors such as moisture and fat content ofthe muscle (MUTSIMOTO et al., 1998) but animal model (oecurrence of mutation on RYR1 gene and muscle metabolic State) should also be controlled in pig experiments (CHEAH et al, 1994(CHEAH et al, , 1995(CHEAH et al, , 1998;;LAHUCKY et al, 1997LAHUCKY et al, , 1998)).From our results on muscle energetic metabolism using 3I P NMR spectroscopy follows that vitamin E may have other biochemical effects not yet investigated.It seems also the effect of vitamin E supplementation on conduetivity and drip loss needs further investigation.

Conclusion
Dietary vitamin E (200 mg/kg diet) administered for 60 days to finishing pigs may have beneficial effects on muscle energetic metabolism, electrical conduetivity and drip loss values but results could depend on the muscle studied and conditions of the experiment (animal model).In pigs oecurrence of mutation in ryanodine reeeptor and muscle metabolic state should be controlled.

Table 1 Groups
of supplementation with vitamin E and organic selenium in diet and concentration of vitamin E (atocopherol) in longissimus dorsi muscle (Least Square means (LSM) und Standardfehler (SE) von Phosphorkomponenten (% von Gesamt) im Musculus longissimus (15 min post mortem) von Schweinen spectrum -12 min obtainingof spectra; 2. spectrum -8 min after 1, spectrum Interactions group x Status; group x sex; * significance P<0.05

Table 2
Colour measured as reflectances and amount of thiobarbituric acid (TBA) related Compounds in muscles longissimus dorsi (LD) and psoas major (PM) of pigs during chill storage (Least Square means (LSM) und Standardfehler (SE) der Konzentration von Vitamin E im Musculus longissimus und von Fleischqualitätsparametem im Musculus longissimus und Musculus semimembranosus unter dem Einfluß einer Fütterung mit erhöhten Vitamin E Gehalt)