Determination of myoglobin concentration in blood-perfused tissue. Eur J Appl Physiol

Faculty of Human Sciences, Institute of Human and Social Science, Kanazawa University, Kanazawa, Japan.
Arbeitsphysiologie (Impact Factor: 2.19). 10/2008; 104(1):41-8. DOI: 10.1007/s00421-008-0775-x
Source: PubMed

ABSTRACT The standard method for determining the myoglobin (Mb) concentration in blood-perfused tissue often relies on a simple but clever differencing algorithm of the optical spectra, as proposed by Reynafarje. However, the underlying assumptions of the differencing algorithm do not always lead to an accurate assessment of Mb concentration in blood-perfused tissue. Consequently, the erroneous data becloud the understanding of Mb function and oxygen transport in the cell. The present study has examined the Mb concentration in buffer and blood-perfused mouse heart. In buffer-perfused heart containing no hemoglobin (Hb), the optical differencing method yields a tissue Mb concentration of 0.26 mM. In blood-perfused tissue, the method leads to an overestimation of Mb. However, using the distinct (1)H NMR signals of MbCO and HbCO yields a Mb concentration of 0.26 mM in both buffer- and blood-perfused myocardium. Given the NMR and optical data, a computer simulation analysis has identified some error sources in the optical differencing algorithm and has suggested a simple modification that can improve the Mb determination. Even though the present study has determined a higher Mb concentration than previously reported, it does not alter significantly the equipoise PO(2), the PO(2) where Mb and O(2) contribute equally to the O(2) flux. It also suggests that any Mb increase with exercise training does not necessarily enhance the intracellular O(2) delivery.

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Available from: Ping-Chang Lin, Sep 26, 2015
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    • "These orthologous globins share much of their overall structure (characteristic ''globin fold''), and many key regions are highly conserved [10]. With similar structure and heme binding properties, the optical characteristics of these proteins are similar [11] [12] (Figs. 2 and 3) and these hemoproteins cannot be distinguished by spectroscopy when in solution together [11]. At wavelengths of 500–700 nm, the absorption spectra of HbO 2 and MbO 2 both show twin absorption peaks: 544 and 582 nm for MbO 2 and 542 and 578 nm for HbO 2 . "
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    ABSTRACT: An accurate determination of myoglobin (Mb) oxygen affinity (P50) can be difficult due to hemoglobin (Hb) contamination and autoxidation of Mb to metMb which is incapable of binding oxygen. To reduce Mb autoxidation, P50 is often measured at refrigerated temperatures. However, the temperature dependent shift in Mb oxygen affinity results in a greater oxygen affinity (lower P50) at colder temperatures than occurs at physiological temperature (ca. 37-39 °C) for birds and mammals. Utilizing the temperature dependent pH shift of Tris buffer, we developed novel methods to extract Mb from vertebrate muscle samples and remove Hb contamination while minimizing globin autoxidation. Cow (Bos taurus) muscle tissue (n = 5) was homogenized in buffer to form a Mb solution, and Hb contamination was removed using anion exchange chromatography. A TCS Hemox Blood Analyzer was then used to quickly generate an oxygen dissociation curve for the extracted Mb. The oxygen affinity of extracted bovine Mb was compared to commercially available horse heart Mb. The oxygen affinity of extracted cow Mb (P50 = 3.72 ± 0.16 mmHg) was not statistically different from commercially prepared horse heart Mb (P50 = 3.71 ± 0.10 mmHg). With high yield Mb extraction and fast generation of an oxygen dissociation curve, it was possible to consistently determine Mb P50 under physiologically relevant conditions for endothermic vertebrates.
    Protein Expression and Purification 11/2014; 107. DOI:10.1016/j.pep.2014.11.004 · 1.70 Impact Factor
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    • "This is more important, as luminal-type breast cancer, although generally more favourable in course, does not respond well to conventional chemotherapy (Bhargava et al, 2010). Myoglobin has different known or alleged functions in muscle tissue including short-term O 2 storage and buffering, facilitating O 2 diffusion, scavenging of NO and ROS and also the reverse (peroxidase activity, NO production) and might be involved in FA metabolism (Wittenberg, 1970; Wittenberg et al, 1975; Khan et al, 1998; Flögel et al, 2001, 2004; Ordway and Garry, 2004; Hendgen- Cotta et al, 2008; Masuda et al, 2008). The functions of Mb in nonmuscle tissues are elusive so far. "
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    ABSTRACT: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.
    British Journal of Cancer 06/2010; 102(12):1736-45. DOI:10.1038/sj.bjc.6605702 · 4.84 Impact Factor
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    • " 000g for 30 min at 4 • C . The clear supernatant was decanted into a small glass tube and again equilibrated with carbon monoxide to ensure binding to Mb . The optical density at 538 ( β band ) and 568 nm ( α band ) was used for calculation of both the Hb and the Mb concentration in the muscle tissue as described in a modified Reynafarje method ( Masuda et al . 2008 ) ."
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    ABSTRACT: Although the O(2) gradient regulates O(2) flux from the capillary into the myocyte to meet the energy demands of contracting muscle, intracellular O(2) dynamics during muscle contraction remain unclear. Our hindlimb perfusion model allows the determination of intracellular myoglobin (Mb) saturation ( ) and intracellular oxygen tension of myoglobin ( ) in contracting muscle using near infrared spectroscopy (NIRS). The hindlimb of male Wistar rats was perfused from the abdominal aorta with a well-oxygenated haemoglobin-free Krebs-Henseleit buffer. The deoxygenated Mb ([deoxy-Mb]) signal was monitored by NIRS. Based on the value of [deoxy-Mb], and were calculated, and the time course was evaluated by an exponential function model. Both and started to decrease immediately after the onset of contraction. The steady-state values of and progressively decreased with relative work intensity or muscle oxygen consumption. At the maximal twitch rate, and were 49% and 2.4 mmHg, respectively. Moreover, the rate of release of O(2) from Mb at the onset of contraction increased with muscle oxygen consumption. These results suggest that at the onset of muscle contraction, Mb supplies O(2) during the steep decline in , which expands the O(2) gradient to increase the O(2) flux to meet the increased energy demands.
    Experimental physiology 05/2010; 95(5):630-40. DOI:10.1113/expphysiol.2009.050344 · 2.67 Impact Factor
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