Simultaneous Measurement of Oxygenation in Intracellular and Extracellular Compartments of Lung Microvascular Endothelial Cells

Abstract

A new technique is described for simultaneous determination of intra- and extracellular oxygen concentrations [partial pressure of O(2) (pO(2))] in bovine lung microvascular endothelial cells (BLMVECs) using electron paramagnetic resonance (EPR) oximetry. The measurements were performed in BLMVEC suspensions of a 20- micro l volume containing 4,000 cells. The extracellular pO(2) was measured using a trityl EPR probe [triarylmethyl (TAM), 10 micro M], a tricarboxylate anion radical, that stays exclusively in the extracellular space. The intracellular oxygen was measured using a pre-internalized particulate spin probe, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-naphthalocyanine (LiNc-BuO). Because there is a wide discrepancy in the reported values of cellular oxygenation by and large due to differences in the methods employed, we utilized the dual EPR probe technique to measure the oxygen gradient that apparently exists across the cell membrane. The intra- and extracellular pO(2) values were 139 +/- 2.5 and 157 +/- 3.6 mm Hg, respectively, for cells exposed to room air. A fairly smaller gradient of oxygen was observed in cells exposed to 7.5% oxygen (pO(2) = 57 mm Hg). In summary, this study confirms the feasibility of simultaneous and accurate measurements of intra- and extracellular pO(2) using LiNc-BuO and TAM EPR oximetry probes.

Application of magnetic field over-modulation for improved EPR linewidth measurements using probes with Lorentzian lineshape

“…For example, although a 10-fold difference in signal-to-noise ratio occurred to the experimental spectra (corresponding to 100 times difference in microwave power), accurate linewidth information has been still achieved through curve fitting. These linewidths obtained at different concentrations are consistent with the previously reported values [9,22,24]. Table 1 summarizes the linewidth information of LiNc-BuO at different oxygen concentrations and different microwave power levels.…”

Skeletal myoblasts transplanted in the ischemic myocardium enhance in situ oxygenation and recovery of contractile function

“…SMs were isolated from the hindlimb skeletal muscle biopsies of mice and cultured as described previously (42). The cells were coincubated with a suspension of sonicated OxySpin crystals for 48 h (15,42). We have recently reported that myoblasts labeled with or coincubated in the presence of OxySpin probes did not show any significant change in viability, metabolism, or proliferation rate (42 …”

“…The particulate probes measure PO 2 in the tissue milieu. Lithium octa-n-butoxy-substituted naphthalocyanine radical (LiNc-BuO) is a particulate oximetry spin probe that we have recently synthesized and validated for in vivo oximetry (2,15,27,42,43). The LiNc-BuO crystals are composed of stacks of neutral radicals of lithiated naphthalocyanine macrocycles (28).…”

“…The EPR spectra of these particulates are characterized by a single and very narrow absorption peak due to the strong exchange coupling between the unpaired electrons within the molecular stack. The probe, in the form of submicron-sized (270 Ϯ 120 nm) crystals (hereafter referred to as OxySpin), can be internalized in cells without compromising cell integrity and function (15,27). A unique advantage of this probe is that it is retained in cells/tissues for substantially long periods, thus enabling continuous monitoring of PO 2 for several months and possibly longer (27).…”

Cardioprotection by HO-4038, a novel verapamil derivative, targeted against ischemia and reperfusion-mediated acute myocardial infarction

“…Once implanted, the probe responds to PO 2 in the immediate surrounding, mostly at the crystal-tissue interface, enabling magnetic resonance-based noninvasive and repeated measurements of PO 2 for a prolonged period from the site of implantation. The technology has been well validated for measurements of PO 2 from single cells to whole organs (16,26,38).…”