J. Anim. Sci. 2004. 82:2742-2749
© 2004 American Society of Animal Science
Effect of supplemental vitamin D3 concentration on concentrations of calcium, phosphorus, and magnesium relative to protein in subcellular components of the longissimus and the distribution of calcium within longissimus muscle of beef steers1,2
J. L. Montgomery*,3,
J. R. Blanton, Jr.*,
R. L. Horst
,
M. L. Galyean*,
K. J. Morrow, Jr.¶,4,
V. G. Allen
,
D. B. Wester
and
M. F. Miller*,5
* Department of Animal and Food Science,
and
Department of Range, Wildlife, and Fisheries Management, and
and
Department of Plant and Soil Sciences, Texas Tech University, Lubbock 79409;
and
National Animal Disease Center, USDA-ARS, Ames, IA 50010; and
and
¶ Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock 79409
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Abstract
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The effect of supplementing diets with various levels of vitamin D3 to provide 0, 0.5, 1, and 5 million IU/(steer•d) for 8 d before slaughter on the mineral content and localization of Ca in LM and muscle fragments was studied during the postmortem aging process. Twelve feedlot steers of three biological types were given access to the four levels of vitamin D for 8 d before slaughter. Differential centrifugation techniques were used to determine the concentrations of minerals relative to protein in different muscle fragments on d 3 and 21 postmortem. Electron microscopy visualization of bound Ca indicated that vitamin D3 mobilized Ca from the sarcoplasmic reticulum and transverse tubule system into the myofibrils. Bound Ca was concentrated near the Z-line at the A-band/I-band juncture within the sarcomere. Supplementing steers with 1 and 5 million IU/(steer•d) of vitamin D3 increased (P < 0.05) Ca, P, and Mg concentrations per unit of protein in the cytosol. Soluble cytosolic Ca concentrations were greater (P < 0.05) on d 21 than on d 3 postmortem only when steers were supplemented with 5 million IU/d. Concentrations of Ca, P, and Mg in isolated tissues were increased (P < 0.05) in nuclei and myofibrilar proteins by supplementing steers with 1 and 5 million IU/(steer•d) of vitamin D3. All supplemental vitamin D3 treatments also increased (P < 0.001) Mg concentrations in the cytosol, regardless of aging treatment, and increased Mg concentrations (P < 0.04) within the mitochondria at d 3 postmortem. Thus, supplementation of feedlot steers with vitamin D3 at levels of 0.5 to 5 million IU/(steer•d) increased Ca concentrations within respiring muscle, resulting in increased bound tissue Ca concentrations. When the respiring muscle was converted to meat, the increased bound tissue Ca resulting from vitamin D3 treatment released Ca concentrations into the cytosol during aging (P < 0.05). Results of this study indicate that vitamin D3 supplementation increased total cytosolic Ca, P, and Mg concentrations in meat.
Key Words: Aging • Beef • Calcium • Minerals • Postmortem • Vitamin D
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Introduction
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Because of the link between Ca and tenderness, a number of studies have been conducted to increase muscle Ca concentrations. Carcasses have been infused with CaCl2 solutions to activate the calpain proteases to improve tenderness (Koohmaraie et al., 1988
, 1989, 1990). Meat also has been injected with CaCl2, resulting in enhanced tenderness (Wheeler et al., 1993, 1997; Kerth et al., 1995). More recently, research has shown that meat tenderness can be increased by supplementing cattle finishing diets with vitamin D3 (VITD) by mobilizing Ca (Swanek et al., 1999; Montgomery et al., 2000, 2002).
Muscle is a target organ for VITD (Boland, 1986). Electron microscopy studies and electrophoretic measurements have demonstrated that VITD deficiency results in degenerative muscle, with a loss of troponin-C and actin (George et al., 1981; de Boland et al., 1983). Toury et al. (1990) reported that vitamin D repletion of rats increased the concentration of bound Ca to the myofibril and free cytosolic concentrations of Ca and P. This increase in free cytosolic Ca could have stimulated Ca-activated calpains and could be responsible for muscle structural alterations. Thus, muscle weakness from VITD deficiency could be explained by increases in muscle Ca.
In contrast to a deficiency, Swanek et al. (1999) reported that VITD supplementation at 5 million IU/d increased free Ca concentrations in meat; however, effects of VITD supplementation on P and Mg distribution in muscle were not reported. Although VITD treatments seem to increase Ca content in muscle, it remains unclear whether the activation of the calpain system and increased proteolysis are a result of increased cytosolic muscle Ca or from postmortem changes in cytosolic Ca. The objective of our study was to determine the effects of VITD supplementation and postmortem aging on the mineral content of various fractions of the LM of beef steers.
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Materials and Methods
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Animals
Three steers (one of each of the three biological types: Bos indicus, Bos taurus-Continental, and Bos taurus-English) were fed diets containing each of four vitamin D3 concentrations (0, 0.5, 1, and 5 million IU/(steer•d); n = 12) described by Montgomery et al. (2004a,b). Calcium localization was visualized via potassium pyroantimonate staining and electron transmission microscopy. Diets had been supplemented with VITD for 8 d before slaughter to improve beef tenderness, as described by Montgomery et al. (2004a). Mineral analyses were determined on a subsample of six steers of the four individual VITD treatments (n = 24) that were selected to be closest to the mean tissue Ca concentration of each of the respective VITD treatments (Montgomery et al., 2004b). Mineral analyses were measured on LM samples at 3 and 21 d postmortem to determine mineral concentrations of the cytosol and bound mineral concentrations of the isolated cell components.
Electron Microscopy
Five-gram LM samples were taken from the 12 selected animals at 20 min postmortem. Samples were cut longitudinally to form 100- to 200-µm-thick slices, and fixed in a 4% (wt/vol) cold potassium pyroantimonate, 2% (vol/vol) paraformaldehyde, and then 1% (vol/vol) phenol (pH 7.4) solution for 4 h to determine bound Ca localization, according to the procedures of Mentré and Escaig (1988). Pyroantimonate binds to Ca, forming dark granules when visualized using electron microscopy. In addition, a sample from one steer (Bos taurus-English) supplemented with 5 million IU of VITD was treated with the fixation solution plus 2 mM ethylenebis oxyethylenenitrilotetraacetic acid (EGTA), a compound that will solubilize divalent cations, as a negative control. Sections were dehydrated for 10 min through a graded series of ethanol/water solutions (vol/vol) consisting of 50, 75, 85, 95, 95, 100, 100, 100, and 100% ethanol. Once fixed, the samples were visualized on a Hitachi H 60 (Hitachi, Tokoyo, Japan) electron-transmission microscope.
Muscle Fractionation
The fibrous envelope, exterior fat, and connective tissue of the muscles were carefully removed, and 5 g of the muscle fibers were rapidly minced and homogenized for 60 s in 20 mL of 0.25 M sucrose with a Polytron homogenizer (model PT-MR-2100; Brinkmann Instruments, Inc., Westbury, N.Y.). Samples were then strained through a metal strainer (approximately 1-mm pore size) and differentially centrifuged to separate specific fractions. Nuclei, cell debris, and large fragments of the myofibrils were removed by centrifugation at 1,500 x g, small myofibril fragments were sedimented at 3,000 x g, mitochondria at 8,000 x g, and sarcoplasmic reticulum at 180,000 x g, as described by Toury et al. (1990). Each centrifugation tube was rinsed and vortexed twice with 10 mL of 0.25 M sucrose for removal of sedimentary cellular proteins. The supernatant fluid of the last centrifugation (180,000 x g) retained soluble elements only and was considered to represent the isolated cytosol. Protein concentration of each fraction was quantified by the biuret reaction using BSA as the standard (Layne, 1957). Mineral concentrations in the various fractions were expressed per unit of protein.
Muscle Ca, P, and Mg concentrations were measured in the particulate fractions. Samples were first digested using nitric and perchloric acid wet ashing procedures (Muchovej et al., 1986). Minerals were then quantified in duplicate by atomic emission using a Thermo Jarrell Ash Trace Scan (Franklin, MA) inductively coupled plasma spectrophotometer.
Statistical Analyses
Mineral data from the muscle fragments were analyzed as a completely randomized, split-plot design, using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The whole plot included VITD treatment, which was tested using vitamin D treatment nested within animal as an error term. Postmortem aging and the postmortem aging x VITD interaction were the effects included in the subplot, which were tested using the residual error term. Least squares means were computed, and treatment differences were calculated using the LSD method at an alpha level of 0.05. Critical differences were calculated for the LSD by calculating the Saiterthwaite df for the t-values (Saiterthwaite, 1946). Pooled standard errors were calculated according to Steel and Torrie (1980).
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Results
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Visualization of Ca binding to respiring LM samples is presented in Figures 1 through 3. When EGTA was included in the fixative of an animal treated with VITD, the dark-Ca-pyroantimonate granules were not evident (Figure 1). Presumably, the EGTA complexed with free Ca such that pyroantimonate granules were not evident by electron microscopy. Because the addition of EGTA largely eliminated the dark Ca-pyroantimonate granules, it was assumed that the dark Ca-pyroantimonate granules seen in Figures 2 and 3 were not artifacts of fixation or staining. In LM samples from steers fed no supplemental VITD, the Ca-pyroantimonate granules were massed in the transverse-tubule system and in the terminal cisternae system (Figure 2). A small amount of the Ca granules were located near the Z-line. In steers fed 0.5 million IU of VITD (Figure 3), Ca-pyroantimonate granules were massed close to the Z-lines (on either side), and large dark lines were evident at the A- and I-band juncture. Bound Ca deposits also were observed in the interior of the myofibril. Observations at the higher VITD supplementation rates (1 and 5 million IU of VITD) gave similar patterns, except that nonspecific precipitates were more wide spread (data not shown). In general, VITD treatments decreased the quantity of Ca bound within the transverse-tubule system and the sarcoplasmic reticulum, but increased binding in the myofibril near the Z-lines of respiring LM.
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Figure 1. Electron microscopy slide of LM from a steer supplemented with 5 million IU of vitamin D3 for 8 d. Dark precipitates of bound Ca disappeared after the fixation solution was treated with ethylenebis oxyethylenenitrilotetraacetic acid (EGTA; bar = 1 µm).
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Figure 3. Electron microscopy slide of LM from a steer supplemented 0.5 million IU of vitamin D3 for 8 d. Calcium-pyroantimonate granules were massed close to the Z-lines, constituting large dark lines at the junction of the A- and I-bands (bar = 1 µm).
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Figure 2. Electron microscopy slide of LM from a steer that was not supplemented with vitamin D3 (control). Calcium-pyroantimonate granules were concentrated in the T-system and terminal cisternae. A small portion of the calcium-pyroantimonate granules are evident near the Z-lines (bar = 1 µm).
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Concentration of protein did not differ (P > 0.50) among VITD treatments within the various particulate cellular fractions (data not shown), thereby allowing mineral concentrations to be expressed per unit of protein. Supplementing steers with 1 or 5 million IU/(steer•d) of VITD increased (P < 0.05) free cytosolic Ca concentrations at 3 and 21 d postmortem (Table 1); however, free Ca concentrations in the cytosol did not (P = 0.23) increase between 3 and 21 d of aging. When the LM of steers fed 1 and 5 million IU/d was aged 21 d, the amount of nuclei and large myofibril cellular fragment contents of bound Ca were also increased (P < 0.05). Vitamin D3 supplementation did not affect (P > 0.35) Ca concentrations of small sedimented myofibril fragments, mitochondria, or sarcoplasmic reticulum proteins.
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Table 1. The effect of supplementing diets of feedlot steers with 0, 0.5, 1.0, or 5.0 million IU/(steer•d) of vitamin D3 for eight consecutive days before slaughter and postmortem aging on the calcium content of homogenate and subcellular fractions of LM (µg of Ca/mg of protein)
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Steers fed 1 and 5 million IU/d of VITD also had increased (P < 0.001) concentrations of free P within the cytosol at both 3 and 21 d postmortem (Table 2). Furthermore, these two treatments increased (P = 0.001) P concentrations of large myofibril fragments and other larger cellular proteins in samples aged 21 d. Vitamin D3 treatments did not, however, affect (P > 0.07) bound P concentrations of small myofibrils, mitochondria, or sarcoplasmic reticulum proteins.
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Table 2. The effect of supplementing diets of feedlot steers with 0, 0.5, 1.0, or 5.0 million IU/(steer•d) of vitamin D3 for eight consecutive days before slaughter and postmortem aging on the phosphorus content of homogenate and subcellular fractions of LM (µg of P/mg of protein)
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Steers fed supplemental VITD had increased (P < 0.001) concentrations of Mg in the cytosol on both 3 and 21 d postmortem (Table 3) compared with the controls. Steers fed diets that provided 5 million IU/d had the greatest cytosolic concentrations of Mg. All VITD treatments increased (P < 0.05) mitochondrial concentration of Mg per unit of protein at 3 d postmortem, whereas steers supplemented with 0.5 million IU/d of VITD maintained increased (P < 0.05) concentrations of mitochondrial Mg at 21 d postmortem. Steers supplemented with 1 and 5 million IU of VITD daily had more (P < 0.05) Mg in large myofibril fragment proteins than did unsupplemented steers at 21 d postmortem but not at 3 d postmortem. Vitamin D3 treatments did not affect (P > 0.22) Mg concentrations of small sedimented myofibrils or sarcoplasmic reticulum components.
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Table 3. The effect of supplementing diets of feedlot steers with 0, 0.5, 1.0, or 5.0 million IU/(steer•d) of vitamin D3 for eight consecutive days before slaughter and postmortem aging on the magnesium content of homogenate and subcellular fractions of LM (µg of Mg/mg of protein)
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In the respiring muscle, collected 20 min postmortem, VITD supplementation increased concentrations of Ca near the Z-line, which in turn affected cytosolic Ca concentrations during postmortem aging. Supplementation increased the concentrations of free cytosolic Ca, P, and Mg. When steers were fed 5 million IU/d of VITD, concentrations of free cytosolic minerals tended to be greater at 21 d than at 3 d postmortem.
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Discussion
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Toury et al. (1990) demonstrated that repletion of VITD-deficient rats doubled Ca concentrations in the cytosol of skeletal muscle. Results of the current study demonstrated that VITD supplementation could increase free Ca by 2.6-fold at 3 d postmortem, with a 6-fold increase at 21 d postmortem. Taylor et al. (1995) reported that extracellular Ca leaked into the muscle cell during the conversion of muscle to meat. Cytosolic Ca also increases in muscle in response to decreasing pH as muscle enters rigor mortis (El-Saleh and Solaro, 1988; Gulati and Babu, 1989; Parsons et al., 1997) as a result of release from cellular organs and binding to proteins. Jeacocke (1993) showed that intracellular free Ca concentrations are tightly maintained between 0.1 and 0.2 µM in the live muscle cells, but both Ca and Mg concentrations increase within the cytosol as muscle enters rigor. Moreover, both Mg and Ca concentrations increase dramatically with the induction of rigor (Burton, 1983). In respiring muscle, the concentrations of free Ca and Mg also vary with muscle contraction, and approximately 50% of Mg is bound, whereas the remaining half is "free" (Somlyo and Somlyo, 1981). Results of the current study provide evidence to support the belief that cytosolic Ca and Mg concentrations increase as a consequence of postmortem aging; however, the major increase in concentrations of these cations occurs shortly after death and rigor.
Magnesium in muscle greatly decreases the rate of Ca binding to sites that bind Mg and Ca competitively, as found in troponin, parvalbumin, and myosin (Potter et al., 1981; Stephenson, 1981). This action would consequently lead to an increase in free cytosolic Ca as a result of VITD supplementation and increased Ca bound near the Z-line. Furthermore, VITD supplementation increases the amount of bound Ca in the muscle cell before slaughter and spurs development of rigor mortis. Thus, the induction of rigor seems to result in a process whereby cytosolic Ca concentrations may increase more quickly and to a greater extent in VITD-supplemented steers than in control steers. Increased concentrations of Ca in the initial periods of postmortem storage can activate the calpains to a greater extent, resulting in increased myofibrillar proteolysis (Edmunds et al., 1991; Boehm et al., 1998).
Phosphorus is a cellular activator, and increased binding of P to the muscle cell surface is probably a result of the increased receptor activation from VITD supplementation. The observed increase in cytosolic Mg in VITD-supplemented steers may also have increased DNA synthesis (Touyz and Yao, 2003), calpain production (Saatman et al., 2001), and Ca-binding protein production (Hemmingsen, 2000; Ritchie et al., 2001). Additionally, there is some difficulty in interpreting the ion-binding results because these cations may have been redistributed during the subcellular fractionation process. Homogenization disrupts the sarcoplasmic reticulum, releasing any free or unbound cations, primarily Ca. Additionally, without ATP present, the Ca will not accumulate in the sarcoplasmic reticulum, which is partially why the electron micorosocpy of the current study was collected on respiring tissue. Thus, the higher concentrations of Ca in the cytosol likely reflect, to some degree, a greater concentration of binding agents such as nucleotides that will be free in this pool.
Our results demonstrated that concentrations of Ca, P, and Mg relative to protein in various cellular fractions increased within meat as a result of VITD supplementation and postmortem aging. Anthony et al. (1986) reported that VITD supplementation of rats resulted in increased muscle deposition of Al, Ca, and K. Mitochondria have been shown to be a binding storehouse for Ca (Greaser, 1977). In the current study, however, Mg, but not Ca, binding was increased in mitochondria as a result of VITD treatment.
Yarom and Meiri (1972) and McCallister and Hadek (1973) were the first to report that pyroantimonate precipitates indicated Ca-binding sites within skeletal muscle. In their samples, Ca-binding was only near the Z-lines, and it was less dramatic than in the current study. The use of osmium within the fixation step also has been suggested to improve electron microscopy transmission, but visualization was noted as problematic with unrepeatable results by Locker and Wild (1984). Binding Ca to structural myofibrillar proteins is another mechanism that helps maintain steady-state cytosolic concentrations of Ca. Wróblewski and Edström (1994) found that Ca concentrations were highest in the Z-line and A-band/I-band juncture, the location of actin, myosin, titin, and nebulin molecules. The binding of Ca in the electron-microscopy slides in the current study may be a result of Ca binding to a number of proteins located in or near the A-band/I-band juncture that have Ca-binding affinities, such as troponin C, titin, nebulin, actin, and/or myosin (Sjodin, 1982; Tatsumi et al., 1997). Labeit et al. (2003) demonstrated Ca binding to the praline-, glutamine-, valine-, and lysine (PEVK)-rich region of titin, which would be consistent with the electron microscopy visualization of Ca in the current study. Within muscle cells, the calpains are localized with myofibrils, mitochondria, and nuclei. In skeletal muscle myofibrils, µ- and m-calpains and Ca, the essential activator of the calpains, all have a similar localization and are most densely located near the Z-line (Taylor et al., 1995). The endogenous inhibitor of the calpains, calpastatin, seems to be localized with both µ-calpain and m-calpain (Goll et al., 1992).
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Implications
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The mechanism by which vitamin D3 supplementation affects tenderness seems to involve mobilization of Ca and an increase in Ca concentration near the Z-line and A-band/I-band juncture in live muscle. Our results indicate that VITD supplementation and postmortem aging both dramatically affect the ratio of Ca, P, and Mg concentrations to protein in muscle and meat. Feeding vitamin D3 at 0.5 to 5 million IU/(steer•d) increased the ratio of Ca in cellular components in muscle, which was then released during postmortem aging and rigor development, increasing the ratio of Ca to protein in cytosol. Improvements in meat tenderness with vitamin D3 supplementation might be explained by an increase in the Ca content within the respiring muscle cell and activation of the calpain system in respiring muscle, resulting in further myofibrillar proteolysis.
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Footnotes
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1 This research was funded in part by a grant from the National Cattlemen’s Beef Association, Texas Beef Council, Roche Vitamins, Inc., and the Center for Feed Industry Research and Educations at Texas Tech University. 
2 Texas Tech Univ. Publ. No. T-5-451. The authors thank M. C. Hastert of the Texas Tech University Health Science Center for her help in the electron microscopy work and P. Brown for his help and guidance with the mineral composition work.
3 Current address: Intervet Inc., Millsboro, DE 19966.
4 Current address: Meridian Bioscience, Inc., Cincinnati, OH 45244.
5 Correspondence: Box 42162 (phone: 806-742-2804; fax: 806-742-0169; e-mail: mfmrraider{at}aol.com).
Received for publication October 7, 2003.
Accepted for publication May 27, 2004.
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Abstract
Introduction
