Rapid, simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins

Abstract

A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.

A comparison of bleeding efficiency, microbiological quality and lipid oxidation in goats subjected to conscious halal slaughter and slaughter following minimal anesthesia

“…For every sample, 5 g of pulverised muscle tissue were mixed with 15 mL of ice cold extraction buffer containing 80 mM KCl and 50 mM Tris-HCL at pH 8.0 and homogenised (Wiggen Hauser, Germany) for 40 s. Ice cold extraction buffer was used to prevent denaturation of myoglobin by acid produced during glycolysis whereas KCl mimics intracellular ion concentration (O'Brien et al, 1992). The homogenates were rinsed with additional 5 mL of the buffer and centrifuged at 5000 g for 10 min at 21 ºC using a refrigerated centrifuge machine (Avanti® J-26 XPI, Beckman Coulter®, USA) to clarify the supernatant .…”

Analysis of changes in reflectance measurements on biological tissues subjected to different probe pressures

“…Note that the dominant contributors to the optical absorption in muscle come from hemoglobin and myoglobin. The absorption spectrum of myoglobin is very similar to that of hemoglobin and its concentration in muscles is typically lower than that of hemoglobin, 14,15 therefore its contribution to the optical absorption was neglected in this paper. Although the blood volume is reduced when the blood vessels are compressed, the concentration of myoglobin might increase, counteracting the decrease in hemoglobin absorption.…”

Calibrated histochemistry applied to oxygen supply and demand in hypertrophied rat myocardium

“…Fixed sections, 10 m thick, were incubated in a medium consisting of 760 l water, 40 l ethanol containing 50 mM ortho-tolidine, and 20 l of 1 M tertiarybutyl-hydroperoxide (O'Brien et al, 1992) for 60 min at 22-25°C. The absorbance of 40 myocytes in the section was determined as described above at 436 nm.…”