Maltose-mediated long-term stabilization of freeze- and spray- dried forms of bovine and porcine hemoglobin

Abstract

Slaughterhouse blood represents a valuable source of hemoglobin, which can be used in the production of heme-iron based supplements for the prevention/treatment of iron-deficiency anemia. In order to obtain a stable solid-state formulation, the effect of maltose addition (30 %) on the stability and storage of bovine and porcine hemoglobin in powders obtained by sprayand freeze-drying (without maltose: Hb; with maltose: HbM) were investigated. Differential scanning calorimetry of spray-and freeze-dried powders indicated satisfying quality of the formulation prepared with maltose on dissolving back into solution. After two-year storage at room temperature (20±5 °C) in solid forms, protected from moisture and light, rehydrated spray-and freeze--dried HbM were red, while Hb were brown. Dynamic light scattering showed the presence of native hemoglobin monomers in rehydrated spray-and freeze--dried HbM, but their agglomerates in Hb samples. UV-Vis spectrophotometry confirmed an absence of significant hemoglobin denaturation and methemoglobin formation in HbM freeze-dried powders. In spray-dried HbM, an increased level of methemoglobin was detected. The results confirmed the stabilizing effect of maltose, and suggested its use in the production of long-term stable solid-state formulations of hemoglobin, along with drying processes optimization.

Modulation of Functional Characteristics of Mesenchymal Stromal Cells by Acellular Preparation of Porcine Hemoglobin

“…After gradual hemolysis, the membranes of lysed erythrocytes were precipitated by centrifugation, at 4 • C, at 3200× g for 40 min and washed with PBS three times. The supernatants were further purified by tangential ultrafiltration through 0.2 µm and 100 kDa pore size filters (Viva Flow ® 50, Sartorius AG, Göttingen, Germany) [18]. Acellular porcine hemoglobin preparation was characterized by means of UV-Vis spectroscopy, photon correlation spectroscopy, SDS-PAGE, and isoelectric focusing, as reported in Drvenica et al [18] and Stančić et al [16].…”

“…The supernatants were further purified by tangential ultrafiltration through 0.2 µm and 100 kDa pore size filters (Viva Flow ® 50, Sartorius AG, Göttingen, Germany) [18]. Acellular porcine hemoglobin preparation was characterized by means of UV-Vis spectroscopy, photon correlation spectroscopy, SDS-PAGE, and isoelectric focusing, as reported in Drvenica et al [18] and Stančić et al [16]. Purified hemoglobin samples were collected at −20 • C, and the tests on cell cultures were completed for less than two years from hemoglobin isolation, since hemoglobin remains a native protein during this period [18].…”

“…Acellular porcine hemoglobin preparation was characterized by means of UV-Vis spectroscopy, photon correlation spectroscopy, SDS-PAGE, and isoelectric focusing, as reported in Drvenica et al [18] and Stančić et al [16]. Purified hemoglobin samples were collected at −20 • C, and the tests on cell cultures were completed for less than two years from hemoglobin isolation, since hemoglobin remains a native protein during this period [18]. Additionally, by using the ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) test, we confirmed the ability of the porcine hemoglobin preparation to reduce ABTS radicals in a dose-dependent manner, indicating preserved divalent iron in heme (Supplementary Material, Figure S1).…”

Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

“…The hemoglobin used in this study was isolated from porcine and bovine slaughterhouse blood by the process our group had optimized, involving gradual hypotonic hemolysis, as previously described [15]. Supernatants containing hemoglobin were purified by tangential microfiltration and the protein and lipid contents were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) and isoelectric focusing [16], and by thin-layer chromatography (TLC) and gas chromatography [17], respectively. Since UV-Vis spectra and dynamic light scattering analysis showed the absorption bands characteristic for native oxyhemoglobin, and as there were no significant individual variations between hemoglobin samples isolated from different animals of the same species [16], pooled hemoglobin samples were used for the cell culture experiments.…”

Insight into the Biological Activity of Hennosides—Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead

“…The hennosides A, B and C in the concentration range between 1 ng/mL–1 mg/mL were incubated with outdated bovine hemoglobin (bHb) (erythrocytes derived from cattle slaughterhouse, as described by Drvenica et al [ 36 ], dissolved in 0.9% NaCl or 0.9 NaCl containing 10% FCS (NaCl-FCS). After the incubation period of 90 min at 37 °C, the OD 630 value, which is the methemoglobin characteristic absorption peak, was obtained on microplate reader.…”