Myoglobin Oxygenation Research Articles

The effect of pH and aging on mitochondrial reduction of bovine myoglobin's affinity for oxygen

In skeletal muscles, mitochondria have been shown to decrease the oxygen affinity of myoglobin. In this study, we investigated whether the mitochondrial function of decreasing myoglobin affinity for oxygen persists and operates at the final pH of postmortem bovine skeletal muscle. The oxygen affinity and myoglobin consumption in the presence of mitochondria obtained from fresh and wet-aged beef were evaluated and compared at pH 5.1, 5.6, and 5.7. The results showed that mitochondria obtained from fresh beef preserved myoglobin oxygen consumption and affinity interference, whereas those obtained from wet-aged beef did not. Oxygen consumption and affinity interference were mostly absent at pH 5.1 and were higher at pH 5.7 than those at pH 5.6. Our findings suggest that mitochondria contribute both to an increase in the oxygen affinity of myoglobin in aged meat and a decrease in the oxygen affinity of myoglobin in high-pH meat, such as dark-cutting beef.

Effects of low-dose sodium nitrite on the structure of yak meat myoglobin during wet curing.

The effects of low doses of sodium nitrite on yak meat colouring, myoglobin oxygenation status, myoglobin aggregation and myoglobin structure were evaluated using Fourier transform infrared spectroscopy, laser micro-Raman spectroscopy and liquid chromatography-electrospray ionization tandem mass spectrometry. The results showed that the yak meat redness value increased steadily relative to that of the control after the addition of low dose sodium nitrite. The nitrosomyoglobin level gradually increased and was significantly higher in the sodium nitrite-treated group than in the control group. The secondary structures were also transformed. The Cα-N bond extended and then contracted, the area of the haem core decreased and then increased and the frequency of contraction increased. A total of 34 nitrosylated peptides were identified, of which 15 were stable and 19 were unstable. These findings show that low doses of sodium nitrite facilitated the dynamic transformation of the myoglobin nitrosylated peptide fragment, which in turn preserved the colour of the meat.

Effect of Blood Flow on Hemoglobin and Myoglobin Oxygenation in Contracting Muscle Using Near-Infrared Spectroscopy.

Insufficient O2 delivery to, and uptake by skeletal muscle can produce mobility limitations for patients with chronic diseases. Near-infrared spectroscopy (NIRS) can be used to noninvasively quantify the balance between skeletal muscle O2 delivery and utilization during contraction. However, it is not clear how the oxygenated or deoxygenated NIRS signal should be used to assess muscle O2 changes. This issue is related to the fact that the contributions of hemoglobin (Hb) and myoglobin (Mb) cannot be distinguished. This conundrum can be resolved by quantitative analysis of experimental data by computer simulations with a mechanistic, mathematical model. Model simulations distinguish dynamic responses of the oxygenated (HbO2, MbO2) and deoxygenated (HHb, HMb) contributions to the NIRS signal components (HbMbO2, HHbMb). Simulations of muscle O2 uptake and NIRS kinetics correspond closely to published experimental data (Hernández et al.,J Appl Physiol 108: 1169-1176, 2010). Simulated muscle O2 uptake and oxygenation kinetics with different blood flows indicate (1) faster O2 delivery is responsible for slower muscle oxygenation kinetics; (2) Hb and Mb contributions to the HbMbO2 are similar (40-60%); and (3) Hb and Mb contributions to the HHbMb are significantly different, 80% and 20%, respectively. The effect of slow blood flow kinetics on oxygenated Hb and Mb contributions is minimal. However, the effect on the imbalance between O2 delivery and utilization rates causes significant overshoots and undershoots of deoxygenated Hb and Mb contributions. Model analysis in combination with NIRS measurements and information on hemodynamic and microvascular distribution can help to determine the use of NIRS signal in evaluating the factors limiting exercise tolerance in health and disease states.

Near-Infrared Spectroscopy (NIRS) of Muscle HbO2 and MbO2 Desaturation During Exercise.

Continuous noninvasive monitoring of muscle oxygenation has important clinical applications for muscle disorders such as compartmentation syndrome, fibromyalgia, deep vein thrombosis, malignant hyperthermia, and the assessment of training in athletic performance. NIRS has precisely such potential and has been used to detect deep venous thrombosis, evaluate athletic performance, and assess limb reperfusion and revascularization. The aim of this study was to examine the relationship between muscle hemoglobin oxygen (HbO2) and myoglobin (MbO2) desaturation using NIRS combined with venous blood sampling and HbO2 desaturation during forearm muscle exercise. Eleven normal subjects were studied, with informed consent and an IRB-approved protocol. A NIRS sensor (INVOS4100, Somanetics, Corp.) was applied on the volar aspect of the forearm. The subjects exercised their forearm by clenching and relaxing their fist while observing the oximeter and driving the reading to specified levels from 90% to 15% (minimum possible reading). Venous blood samples were withdrawn for measurement of blood gases and oxygen saturation (IL-Co-Oximeter). RSO2 (%) vs VO2 Sat showed a two-component HbO2 desaturation suggesting representation of venous HbO2 desaturation and perhaps myoglobin oxygen (MBO2) desaturation. Subtraction of the linear venous HbO2 curve from the two-component curve suggests MbO2 desaturation at venous hemoglobin oxygen saturation of about 10-20%. Conclusions: The kinetics of desaturation during exercise revealed two components representing HbO2 and MbO2 deoxygenation. The data show that MbO2 represents approximately 40% of the NIRS signal and the balance or 60% to HbO2.

Changes in glycolytic and mitochondrial protein profiles regulates postmortem muscle acidification and oxygen consumption in dark-cutting beef

Dark-cutting beef is a condition in which beef fails to have a characteristic bright-red color when the cut surface is exposed to oxygen. However, the mechanistic basis for this occurrence is not clear. Protein expression profiles were compared between dark-cutting and normal-pH beef using LC-MS/MS-based proteomics. Mass spectrometry analysis identified 1162 proteins in the proteomes of dark-cutting and normal-pH beef. Of these, 92 proteins had significant changes in protein abundance between dark-cutting versus normal-pH beef. In dark-cutting beef, 25 proteins were down-regulated, including enzymes related to glycogen metabolism, glucose homeostasis, denovo synthesis of adenosine monophosphate (AMP), and glycogen phosphorylase activity. In comparison, 27 proteins were up-regulated in dark-cutting beef related to oxidation-reduction processes, muscle contraction, and oxidative phosphorylation. Down-regulation of glycogenolytic proteins suggests decreased glycogen mobilization and utilization, while the up-regulation of mitochondrial transport chain proteins indicates a greater capacity to support mitochondrial respiration in dark-cutting beef. These results showed that changes in proteins involved in glycogenolysis and mitochondrial electron transport would promote the development of high-pH and greater oxygen consumption, respectively; thus limiting myoglobin oxygenation in dark-cutting beef. SignificanceThe current understanding indicates that defective glycolysis causes less carbon flow, leading to less postmortem lactic acid formation and elevated muscle pH in dark-cutting beef. However, to the best of our knowledge, limited research has evaluated how changes in glycolytic and mitochondrial protein abundance regulate postmortem muscle acidification and oxygen consumption in dark-cutting beef. We utilized a shotgun proteomics approach to elucidate potential differences in protein profiles between dark-cutting versus normal-pH beef that may influence differences in postmortem metabolism and muscle surface color characteristics. Our study shows that down-regulation of glycolgenolytic and IMP/AMP biosynthetic proteins results in elevated postmortem muscle pH in dark-cutting beef. In addition, the up-regulation of mitochondrial protein content coupled with the higher muscle pH are conducive factors for enhanced oxygen consumption and less myoglobin oxygenation, contributing to a dark meat color typically associated with dark-cutting beef.

Abstract 409: Investigating the Role of the Mitochondrial Calcium Uniporter (mcu) in Calcium Regulation of Cardiac Mitochondria Using Transmural Spectroscopy and an Integrating Sphere

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-2-AM at 30°C and then perfused at 37°C to washout uncleaved dye. We determined that rhod-2 localized primarily to the mitochondria under our loading conditions, as shown by loading in a heart expressing a GFP-tagged mitochondrial outer membrane protein and analyzing heart slices under super resolution microscopy. Further, addition of Mn 2+ , which quenches cytosolic rhod-2, has little effect on rhod-2 signal. We insert an optical catheter with both white light and 532nm laser into the left ventricle and interleaved the collection of spectra from both light sources. The perfused heart is center mounted in an integrating sphere for spectra collection. The light passes through the ventricle and reflects off the integrating sphere resulting in a near uniform sampling of the transmitted light. Spectral properties from both light sources are determined using a rapid scanning spectrophotometer. Myoglobin oxygenation, cytochrome redox state, and rhod-2 loading are determined by white light absorbance. Ca 2+ bound rhod-2 emission is determined by removing background tissue effects from laser emission spectra and normalizing to tissue absorbance. Using this method we are able to measure changes in Ca 2+ and cytochromes during treatments such as isoproterenol and ischemia-reperfusion. The use of an integrating sphere transmural spectroscopy provides us an unique method to study mitochondrial Ca 2+ signaling in perfused mouse heart loaded with Rhod-2.

Impact of myoglobin oxygenation state prior to frozen storage on color stability of thawed beef steaks through retail display

Consumers consider beef color to be an indicator of freshness and therefore it is a major factor when purchasing beef. The ideal conditions for maintaining color throughout retail display following frozen storage have yet to be well established. Therefore, the objective of this study was to determine the effects of myoglobin oxygenation levels (especially high oxygenation) during freezing on color stability of thawed steaks during retail display (RD) and to determine the impact that frozen storage duration, aging, and packaging films have on meat color after thawing under RD conditions. USDA Choice strip loins (n = 36) were aged for 4 or 20 d. Steaks were randomly assigned to a myoglobin oxygenation level [deoxymyoglobin (DeOxy; packaged within 3 min), oxygenation (Oxy; oxygenated in air for 30 min), or high oxygenation (HiOxy; packaged for 24 h in 80% O2)]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (−10 °C). Following either 0, 2, 4, or 6 months of frozen storage at -5 °C, steaks were removed from the packaging and immediately placed under simulated RD conditions for 7 d. During RD, instrumental color and subjective color were measured every day after the initial 24 h thaw period. Steaks were analyzed for instrumental color (L*, a*, b*), a*:b* ratio, percentage oxymyoglobin, metmyoglobin, and deoxymyoglobin, delta E, redness ratio, subjective discoloration, and lipid oxidation. For all days of RD, steaks that were frozen for 0 months had higher a* values (greater redness) than steaks frozen for 6 months which typically had the lowest a* values (P < .0001). HiOxy steaks frozen for 6 months had the lowest amounts of percentage oxymyoglobin than all other frozen storage periods and myoglobin oxygenation levels on days 4–7 of RD (P < .05). The HiOxy steaks frozen for 4 and 6 months had higher percentage metmyoglobin than DeOxy and Oxy, regardless of packaging (P < .05). Delta E, discoloration, and lipid oxidation were greatest for HiOxy steaks compared to Oxy and DeOxy (P < .05). Extended storage brought about detrimental color effects for all differing levels of myoglobin oxygenation. The HiOxy steaks through the first few days of RD and frozen for under 6 months provided had bright cherry red color, similar to that of DeOxy and Oxy. However, with extended frozen storage and RD, HiOxy steaks had worse color characteristics (more discoloration) than the other myoglobin oxygenation levels.

Impact of myoglobin oxygenation level on color stability of frozen beef steaks.

The emerging market of frozen meat emphasizes the need to better understand beef surface discoloration and the ideal parameters of freezing beef to retain an acceptable color. The objectives of this study were to determine the impacts of myoglobin oxygenation level prior to freezing and frozen storage duration on frozen beef color. USDA Choice strip loins (n = 36) were aged for 4 d or 20 d. Steaks were randomly assigned to a myoglobin oxygenation level [deoxygenated (DeOxy; immediately packaged after cutting), oxygenated (Oxy; oxygenated in air for 30 min), or highly oxygenated (HiOxy; packaged for 24 h in 80% O2)]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (-5 °C). Following either 0, 2, 4, or 6 mo of frozen storage, steaks were removed from the packaging and immediately analyzed for instrumental color (L*, a*, and b*), percent oxymyoglobin, metmyoglobin, and deoxymyoglobin, delta E, redness ratio, a*:b* ratio, hue angle, subjective discoloration, and lipid oxidation. The HiOxy steaks had greater oxygen penetration and the greatest a* values compared with DeOxy and Oxy steaks, regardless of packaging (P < 0.0005). With 4 d of aging, HiOxy steaks had greater a* values than DeOxy and Oxy at all storage times (P = 0.0118). The HiOxy steaks aged for 20 d and frozen for 6 mo had significantly higher delta E values than all other myoglobin oxygenation levels and postmortem aging periods (P < 0.0001). Redness and percent oxymyoglobin were highest for HiOxy steaks within each storage period (P < 0.0002). The HiOxy steaks had the highest percent oxymyoglobin and DeOxy had the lowest percent oxymyoglobin within each aging and storage period (P < 0.01). Conversely, DeOxy steaks had the highest percent metmyoglobin and HiOxy had the lowest percent metmyoglobin when packaged in impermeable film (P < 0.0001). The HiOxy steaks from 20 d of aging had the highest discoloration compared with 4 d aging and more discoloration than all other myoglobin treatments at 6 mo of storage (P < 0.0001). The HiOxy 20 d aged steaks exhibited the highest lipid oxidation values at 2, 4, and 6 mo (P = 0.0224) and HiOxy steaks exhibited a brighter and deeper cherry red color compared with the DeOxy steaks. The HiOxy steaks were greater in redness or similar when compared with Oxy steaks, but experienced more detrimental effects when frozen storage was extended.

Biomolecular Interactions Governing Fresh Meat Color in Post-mortem Skeletal Muscle: A Review

Appearance is an important sensory property that significantly influences consumers' perceptions of fresh meat quality. Failure to meet consumer expectations can lead to rejection of meat products, concomitant loss in value, and potential production of organic waste. Immediately after animal harvest, skeletal muscle metabolism changes from aerobic to anaerobic. However, anoxic post-mortem muscle is biochemically active, and biomolecular interaction between myoglobin, mitochondria, metabolites, and lipid oxidation determines meat color. This review examines how metabolites and mitochondrial activity can influence myoglobin oxygenation and metmyoglobin reducing activity. Further, the review highlights recent research that has examined myoglobin redox dynamics, sarcoplasmic metabolite changes, and/or post-mortem biochemistry.

Impact of Myoglobin Oxygenation State at Freezing on Color Stability of Frozen Beef

ObjectivesMeat color is the number one factor influencing consumer purchase decisions. The emerging market of frozen meat emphasizes the need to understand beef surface discoloration and the ideal parameters of freezing beef to retain a superior color. Therefore, the objectives of this study were to determine the impacts of oxygenation level and frozen storage duration on frozen beef color.Materials and MethodsUSDA Choice strip loins (n = 36) were aged for 4 d or 20 d. Steaks were randomly assigned to a myoglobin state [deoxymyoglobin (DeOxy; immediately packaged), low oxygenation (LoOxy; oxygenated in air for 30 min), and high oxygenation (HiOxy; packaged for 24 h in 80% O2)]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (–20°C). Following either 0, 2, 4, or 6 mo of frozen storage, steaks were removed from the packaging and immediately analyzed for instrumental color (L*, a*, b*), delta E (magnitude of difference in the L*, a*, b* color space), subjective discoloration, lipid oxidation (via thiobarbituric acid reactive substances- TBARS), oxygen penetration, percent oxymyoglobin, metmyoglobin, and deoxymyoglobin (via spectrometer), and redness (calculated as 630nm/530nm). Data were analyzed using PROC Glimmix procedure in SAS as a split-split-plot with an incomplete block and a 2 × 3 factorial.ResultsHiOxy steaks had greater oxygen penetration and the highest a* values compared to DeOxy and LoOxy steaks regardless of packaging (P &lt; 0.0005). Conversely, DeOxy steaks exhibited the lowest oxygen penetration and a* values regardless of film (P &lt; 0.0005). HiOxy steaks at 4 d had higher a* values than DeOxy and LoOxy at all storage times (P = 0.0118). HiOxy steaks had the highest delta E values compared to DeOxy and LoOxy in permeable packaging and with increasing storage time an increase in delta E for the HiOxy steaks was observed (P = 0.0010).Redness and percent oxymyoglobin were highest for HiOxy steaks within each storage period (P &lt; 0.0002). HiOxy and LoOxy steaks were similar in percent oxymyoglobin when in permeable packaging film. HiOxy steaks had the highest percent oxymyoglobin and DeOxy had the lowest percent oxymyoglobin within each aging and storage period (P &lt; 0.01). Conversely, DeOxy steaks had the highest percent metmyoglobin and HiOxy had the lowest percent metmyoglobin when packaged in impermeable film (P &lt; 0.0001). Lowest percent metmyoglobin values were from the 4 d HiOxy steaks at 2, 4, and 6 mo of storage (P = 0.0188).The HiOxy 20 d steaks had the highest discoloration compared to 4 d aging and more discoloration than all other myoglobin treatments at 6 mo of storage (P &lt; 0.0001). Lipid oxidation increased with storage time (P = 0.0169). HiOxy 20 d aged steaks exhibited the highest TBARS values at 2, 4, and 6 mo (P = 0.0224). HiOxy and LoOxy were similar in discoloration and lipid oxidation except with the HiOxy 20 d (which were less desirable).ConclusionHiOxy steaks exhibit a brighter and deeper cherry red color compared to the DeOxy steaks. HiOxy steaks were superior or similar when compared to LoOxy steaks but displayed more detrimental effects when frozen storage was extended. Based on the results, HiOxy steaks aged for 4 d give a superior red color for extended storage with few unfavorable effects. However, it is not advised to freeze deoxygenated steaks and expect a cherry red color through frozen storage.

Modified Atmospheric Packaging and Antioxidant Enhancement Improves Redness of Atypical Dark-Cutting Beef

ObjectivesDark-cutting carcasses occur when muscle pH does not decrease sufficiently during rigor mortis because of antemortem glycogen depletion. Typical dark-cutting beef have a pH &gt; 6.0, however, atypical dark-cutting (ADC) beef have a pH around 5.7- 5.9 but have a darker lean color similar to dark-cutting beef. Previous studies noted that greater muscle pH in dark-cutting beef increase oxygen consumption and decrease myoglobin oxygenation. However, limited research has determined the biochemical basis of ADC. Therefore, the objectives of this study were to characterize the biochemical basis of ADC beef carcasses and to utilize modified atmosphere packaging (MAP) and rosemary enhancement to improve appearance during retail display.Materials and MethodsStrip loins from ADC and USDA Low Choice (C) carcasses were selected from a commercial beef processing plant and transported to the Food and Agricultural Products Center in Stillwater, OK. Control (C) and ADC steaks (prior to enhancement and packaging) were utilized to measure pH, color, oxygen consumption, and proximate compositions. Loin sections were cut into halves and randomly assigned to combinations of packaging types and enhancement treatment. Packaging included high-oxygen MAP (HiOx-MAP; 80% oxygen and 20% carbon dioxide), carbon monoxide MAP (CO-MAP; 0.4% CO, 69.6% N, and 30% CO2), and PVC. Enhancement includes 0.1% rosemary oleoresin pumped to 10% of loin green weights. Following enhancement and packaging, steaks were displayed under retail conditions for 6 d to measure color changes using a HunterLab spectrophotometer. The data were analyzed using the Mixed Procedure of SAS, and the experiment was replicated 13 times (n = 13).ResultsThere were no differences for initial pH or proximate compositions (P &lt; 0.05) between C and ADC. However, ADC had lower initial lightness (L* values), redness (a* values), and red intensity (chroma) compared with C. Further, ADC had greater (P &lt; 0.05) oxygen consumption than C. There was a significant packaging × enhancement × display time interaction resulted for L*, a*, and chroma. CO-MAP and HiOx-MAP in combination with rosemary enhancement improved (P &lt; 0.05) redness of ADC by 61.7% and 42.3%, respectively, compared with ADC in PVC packaging. Similarly, MAP and enhancement improved lightness (L* values) and chroma compared with ADC in PVC. By Day 6 of display, enhanced ADC steaks packaged in both HiOx-MAP and CO-MAP had similar color parameters to control choice steaks; however, non-enhanced ADC steaks had significantly lower a* (P &lt; 0.0001) and chroma (P &lt; 0.01) values when packaged in HiOx-MAP when compared to both C and enhanced ADC.ConclusionThe results suggest that ADC beef has greater oxygen consumption than C steaks even at similar muscle pH. Use of modified atmospheric packaging in combination with enhancement has the potential to improve surface color of ADC beef.

Perfused murine heart optical transmission spectroscopy using optical catheter and integrating sphere: Effects of ischemia/reperfusion

Tissue transmission optical absorption spectroscopy provides dynamic information on metabolism and function. Murine genetic malleability makes it a major model for heart research. The diminutive size of the mouse heart makes optical transmission studies challenging. Using a perfused murine heart center mounted in an integrating sphere for light collection with a ventricular cavity optical catheter as an internal light source provided an effective method of optical data collection in this model. This approach provided high signal to noise optical spectra which when fit with model spectra provided information on tissue oxygenation and redox state. This technique was applied to the study of cardiac ischemia and ischemia reperfusion which generates extreme heart motion, especially during the ischemic contracture. The integrating sphere reduced motion artifacts associated with a fixed optical pickup and methods were developed to compensate for changes in tissue thickness. During ischemia, rapid decreases in myoglobin oxygenation occurred along with increases in cytochrome reduction levels. Surprisingly, when ischemic contracture occurred, myoglobin remained fully deoxygenated, while the cytochromes became more reduced consistent with a further, and critical, reduction of mitochondrial oxygen tension during ischemic contraction. This optical arrangement is an effective method of monitoring murine heart metabolism.

71 Impact of myoglobin oxygenation state at freezing on color stability of frozen and thawed beef

Abstract Myoglobin, when oxygenated, is bright red. The deoxygenated form (purple color) is unstable and can be oxidized to metmyoglobin (brown color). This research was conducted to determine if meat frozen in the oxygenated state could retain bright red color during frozen storage, and after thawing, during retail display. Steaks from 18 USDA Choice strip loins were randomly assigned to a myoglobin state [deoxymyoglobin (DeOxy; immediately packaged), low oxygenation (LoOxy; oxygenated for 30 minutes), and high oxygenation (HiOxy; packaged for 24 h in 80% O2)], frozen storage packaging method (vacuum packaged in oxygen permeable or impermeable film), and retail display after two months of frozen storage [0 d (frozen) and 1–7 d (thawed)]. Following storage, frozen steaks were analyzed for objective color (L*, a*, b*), subjective discoloration, and percent oxymyoglobin (via spectrometer). After thawing, steaks were evaluated throughout 7 d of retail display in oxygen-permeable film. For frozen steaks, there were no differences in discoloration (P&gt;.05). The HiOxy steaks retained the highest percentage oxymyoglobin and highest (reddest) a* values after storage, regardless of packaging film. The lowest percent oxymyoglobin (PPPPP &lt; .05). These data suggest that steaks frozen for two months in the high oxygenation state retain superior color appearance while frozen and during the first part of retail display. It is unknown if this advantage would be retained during longer frozen storage.

22Effects of myoglobin oxygenation on oxygenation-sensitive cardiovascular magnetic resonance images: an in-vitro study

22Effects of myoglobin oxygenation on oxygenation-sensitive cardiovascular magnetic resonance images: an in-vitro study

Mitochondrial functionality and beef colour: A review of recent research

The bright-red colour of meat that consumers prefer depends on the depth of oxygen diffusion into the tissue and myoglobin oxygenation. Interestingly, both processes are influenced by mitochondrial activity in postmortem muscle. The transition of muscle metabolism from aerobic to anaerobic pathways affects various cellular processes including mitochondrial functionality. Numerous studies report that even with compromised structure, mitochondria continue to influence postmortem beef colour via oxygen consumption and metmyoglobin reducing activity. Hence, an in-depth understanding of the pre- and post-harvest factors affecting mitochondrial functionality can significantly enhance existing knowledge of meat colour and colour stability. Several comprehensive reviews have discussed the biochemical factors affecting meat colour, but there are only a few that have sections on the impact of mitochondria on beef colour. Furthermore, advancement of high-throughput techniques such as metabolomics and proteomics has enabled researchers to elucidate metabolite and protein changes related to mitochondria. Therefore, the objective of this review is to provide an overview on the role of mitochondria in beef colour, with a focus on recent advances in mitochondrial research, oxygen consumption, and metmyoglobin reducing ability. Keywords: Myoglobin, metmyoglobin reducing activity, meat colour, metabolomics, oxygen consumption, proteomics

Impact of Myoglobin Oxygenation State at Freezing on Color Stability of Frozen Beef

ObjectivesMeat color is the number one factor influencing consumer purchase decisions. The emerging market of frozen meat emphasizes the need to understand beef surface discoloration and the ideal parameters of freezing beef to retain a superior color. Therefore, the objectives of this study were to determine the impacts of oxygenation level and frozen storage duration on frozen beef color.Materials and MethodsUSDA Choice strip loins (n = 36) were aged for 4 d or 20 d. Steaks were randomly assigned to a myoglobin state [deoxymyoglobin (DeOxy; immediately packaged), low oxygenation (LoOxy; oxygenated in air for 30 min), and high oxygenation (HiOxy; packaged for 24 h in 80% O2)]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (–20°C). Following either 0, 2, 4, or 6 mo of frozen storage, steaks were removed from the packaging and immediately analyzed for instrumental color (L*, a*, b*), delta E (magnitude of difference in the L*, a*, b* color space), subjective discoloration, lipid oxidation (via thiobarbituric acid reactive substances- TBARS), oxygen penetration, percent oxymyoglobin, metmyoglobin, and deoxymyoglobin (via spectrometer), and redness (calculated as 630nm/530nm). Data were analyzed using PROC Glimmix procedure in SAS as a split-split-plot with an incomplete block and a 2 × 3 factorial.ResultsHiOxy steaks had greater oxygen penetration and the highest a* values compared to DeOxy and LoOxy steaks regardless of packaging (P < 0.0005). Conversely, DeOxy steaks exhibited the lowest oxygen penetration and a* values regardless of film (P < 0.0005). HiOxy steaks at 4 d had higher a* values than DeOxy and LoOxy at all storage times (P = 0.0118). HiOxy steaks had the highest delta E values compared to DeOxy and LoOxy in permeable packaging and with increasing storage time an increase in delta E for the HiOxy steaks was observed (P = 0.0010).Redness and percent oxymyoglobin were highest for HiOxy steaks within each storage period (P < 0.0002). HiOxy and LoOxy steaks were similar in percent oxymyoglobin when in permeable packaging film. HiOxy steaks had the highest percent oxymyoglobin and DeOxy had the lowest percent oxymyoglobin within each aging and storage period (P < 0.01). Conversely, DeOxy steaks had the highest percent metmyoglobin and HiOxy had the lowest percent metmyoglobin when packaged in impermeable film (P < 0.0001). Lowest percent metmyoglobin values were from the 4 d HiOxy steaks at 2, 4, and 6 mo of storage (P = 0.0188).The HiOxy 20 d steaks had the highest discoloration compared to 4 d aging and more discoloration than all other myoglobin treatments at 6 mo of storage (P < 0.0001). Lipid oxidation increased with storage time (P = 0.0169). HiOxy 20 d aged steaks exhibited the highest TBARS values at 2, 4, and 6 mo (P = 0.0224). HiOxy and LoOxy were similar in discoloration and lipid oxidation except with the HiOxy 20 d (which were less desirable).ConclusionHiOxy steaks exhibit a brighter and deeper cherry red color compared to the DeOxy steaks. HiOxy steaks were superior or similar when compared to LoOxy steaks but displayed more detrimental effects when frozen storage was extended. Based on the results, HiOxy steaks aged for 4 d give a superior red color for extended storage with few unfavorable effects. However, it is not advised to freeze deoxygenated steaks and expect a cherry red color through frozen storage.

Modified Atmospheric Packaging and Antioxidant Enhancement Improves Redness of Atypical Dark-Cutting Beef

ObjectivesDark-cutting carcasses occur when muscle pH does not decrease sufficiently during rigor mortis because of antemortem glycogen depletion. Typical dark-cutting beef have a pH > 6.0, however, atypical dark-cutting (ADC) beef have a pH around 5.7- 5.9 but have a darker lean color similar to dark-cutting beef. Previous studies noted that greater muscle pH in dark-cutting beef increase oxygen consumption and decrease myoglobin oxygenation. However, limited research has determined the biochemical basis of ADC. Therefore, the objectives of this study were to characterize the biochemical basis of ADC beef carcasses and to utilize modified atmosphere packaging (MAP) and rosemary enhancement to improve appearance during retail display.Materials and MethodsStrip loins from ADC and USDA Low Choice (C) carcasses were selected from a commercial beef processing plant and transported to the Food and Agricultural Products Center in Stillwater, OK. Control (C) and ADC steaks (prior to enhancement and packaging) were utilized to measure pH, color, oxygen consumption, and proximate compositions. Loin sections were cut into halves and randomly assigned to combinations of packaging types and enhancement treatment. Packaging included high-oxygen MAP (HiOx-MAP; 80% oxygen and 20% carbon dioxide), carbon monoxide MAP (CO-MAP; 0.4% CO, 69.6% N, and 30% CO2), and PVC. Enhancement includes 0.1% rosemary oleoresin pumped to 10% of loin green weights. Following enhancement and packaging, steaks were displayed under retail conditions for 6 d to measure color changes using a HunterLab spectrophotometer. The data were analyzed using the Mixed Procedure of SAS, and the experiment was replicated 13 times (n = 13).ResultsThere were no differences for initial pH or proximate compositions (P < 0.05) between C and ADC. However, ADC had lower initial lightness (L* values), redness (a* values), and red intensity (chroma) compared with C. Further, ADC had greater (P < 0.05) oxygen consumption than C. There was a significant packaging × enhancement × display time interaction resulted for L*, a*, and chroma. CO-MAP and HiOx-MAP in combination with rosemary enhancement improved (P < 0.05) redness of ADC by 61.7% and 42.3%, respectively, compared with ADC in PVC packaging. Similarly, MAP and enhancement improved lightness (L* values) and chroma compared with ADC in PVC. By Day 6 of display, enhanced ADC steaks packaged in both HiOx-MAP and CO-MAP had similar color parameters to control choice steaks; however, non-enhanced ADC steaks had significantly lower a* (P < 0.0001) and chroma (P < 0.01) values when packaged in HiOx-MAP when compared to both C and enhanced ADC.ConclusionThe results suggest that ADC beef has greater oxygen consumption than C steaks even at similar muscle pH. Use of modified atmospheric packaging in combination with enhancement has the potential to improve surface color of ADC beef.

Paradoxical arteriole constriction compromises cytosolic and mitochondrial oxygen delivery in the isolated saline-perfused heart.

The isolated saline-perfused heart is used extensively to study cardiac physiology. Previous isolated heart studies have demonstrated lower tissue oxygenation compared with in vivo hearts based on myoglobin oxygenation and the mitochondrial redox state. These data, consistent with small anoxic regions, suggest that the homeostatic balance between work and oxygen delivery is impaired. We hypothesized that these anoxic regions are caused by inadequate local perfusion due to a paradoxical arteriole constriction generated by a disrupted vasoregulatory network. We tested this hypothesis by applying two exogenous vasodilatory agents, adenosine and cromakalim, to relax vascular tone in an isolated, saline-perfused, working rabbit heart. Oxygenation was monitored using differential optical transmission spectroscopy and full spectral fitting. Increases in coronary flow over control with adenosine (27 ± 4 ml/min) or cromakalim (44 ± 4 ml/min) were associated with proportional spectral changes indicative of myoglobin oxygenation and cytochrome oxidase (COX) oxidation, consistent with a decrease in tissue anoxia. Quantitatively, adenosine decreased deoxymyoglobin optical density (OD) across the wall by 0.053 ± 0.008 OD, whereas the reduced form of COX was decreased by 0.039 ± 0.005 OD. Cromakalim was more potent, decreasing deoxymyoglobin and reducing the level of COX by 0.070 ± 0.019 OD and 0.062 ± 0.019 OD, respectively. These effects were not species specific, as Langendorff-perfused mouse hearts treated with adenosine demonstrated similar changes. These data are consistent with paradoxical arteriole constriction as a major source of regional anoxia during saline heart perfusion. We suggest that the vasoregulatory network is disrupted by the washout of interstitial vasoactive metabolites in vitro. NEW & NOTEWORTHY Regional tissue anoxia is a common finding in the ubiquitous saline-perfused heart but is not found in vivo. Noninvasive optical techniques confirmed the presence of regional anoxia under control conditions and demonstrated that anoxia is diminished using exogenous vasodilators. These data are consistent with active arteriole constriction, occurring despite regional anoxia, generated by a disrupted vasoregulatory network. Washout of interstitial vasoactive metabolites may contribute to the disruption of normal vasoregulatory processes in vitro.

Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.

The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.

Intracardiac light catheter for rapid scanning transmural absorbance spectroscopy of perfused myocardium: measurement of myoglobin oxygenation and mitochondria redox state.

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The custom-designed intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 × 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dual-wavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state.NEW & NOTEWORTHY Here, a novel nondestructive real-time approach for monitoring intrinsic indicators of cardiac metabolism and oxygenation is described using a catheter-based transillumination of the left ventricular free wall together with complete spectral analysis of transmitted light. This approach is a significant improvement in the quality of cardiac optical absorbance spectroscopic metabolic analyses.