Ulrike Kreutzer

Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle.

1H NMR has detected both the deoxygenated proximal histidyl NδH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffing the leg to reduce blood flow elicits the appearance of the deoxyMb signal, which increases in intensity as cellular[Formula: see text] decreases. The deoxyMb signal is detected with a 45-s time resolution and reaches a steady-state level within 5 min of pressure cuffing. Its desaturation kinetics match those observed in the near-infrared spectroscopy (NIRS) experiments, implying that the NIRS signals are actually monitoring Mb desaturation. That interpretation is consistent with the signal intensity and desaturation of the deoxyHb proximal histidyl NδH signal from the β-subunit at 73 parts per million. The experimental results establish the feasibility and methodology to observe the deoxyMb and Hb signals in skeletal muscle, help clarify the origin of the NIRS signal, and set a stage for continuing study of O2regulation in skeletal muscle.1H NMR has detected both the deoxygenated proximal histidyl NdeltaH signals of myoglobin (deoxyMb) and deoxygenated Hb (deoxyHb) from human gastrocnemius muscle. Exercising the muscle or pressure cuffing the leg to reduce blood flow elicits the appearance of the deoxyMb signal, which increases in intensity as cellular PO2 decreases. The deoxyMb signal is detected with a 45-s time resolution and reaches a steady-state level within 5 min of pressure cuffing. Its desaturation kinetics match those observed in the near-infrared spectroscopy (NIRS) experiments, implying that the NIRS signals are actually monitoring Mb desaturation. That interpretation is consistent with the signal intensity and desaturation of the deoxyHb proximal histidyl NdeltaH signal from the beta-subunit at 73 parts per million. The experimental results establish the feasibility and methodology to observe the deoxyMb and Hb signals in skeletal muscle, help clarify the origin of the NIRS signal, and set a stage for continuing study of O2 regulation in skeletal muscle.

Myoglobin and hemoglobin rotational diffusion in the cell

The detection of the 1H NMR signal of myoglobin (Mb) in tissue opens an opportunity to examine its cellular diffusion property, which is central to its purported role in facilitating oxygen transport. In perfused myocardium the field-dependent transverse relaxation analysis of the deoxy Mb proximal histidyl NdeltaH indicates that the Mb rotational correlation time in the cell is only approximately 1.4 times longer than it is in solution. Such a mobility is consistent with the theory that Mb facilitates oxygen diffusion from the sarcoplasm to the mitochondria. The microviscosities of the erythrocyte and myocyte environment are different. The hemoglobin (Hb) rotational correlation time is 2.2 longer in the cell than in solution. Because both the overlapping Hb and Mb signals are visible in vivo, a relaxation-based NMR strategy has been developed to discriminate between them.

Myoglobin translational diffusion in rat myocardium and its implication on intracellular oxygen transport.

Current theory of respiratory control invokes a role of myoglobin (Mb)‐facilitated O2 diffusion in regulating the intracellular O2 flux, provided Mb diffusion can compete effectively with free O2 diffusion. Pulsed‐field gradient NMR methods have now followed gradient‐dependent changes in the distinct 1H NMR γ CH3 Val E11 signal of MbO2 in perfused rat myocardium to obtain the endogenous Mb translational diffusion coefficient (DMb) of 4.24 × 10−7 cm2 s−1 at 22°C. The DMb matches precisely the value predicted by in vivo NMR rotational diffusion measurements of Mb and shows no orientation preference. Given values in the literature for the Kroghs free O2 diffusion coefficient (K0), myocardial Mb concentration and a partial pressure of O2 that half saturates Mb (P50), the analysis yields an equipoise diffusion P  O 2 of 1.77 mmHg, where Mb and free O2 contribute equally to the O2 flux. In the myocardium, Mb‐facilitated O2 diffusion contributes increasingly more than free O2 diffusion when the P  O 2 falls below 1.77 mmHg. In skeletal muscle, the P  O 2 must fall below 5.72 mmHg. Altering the Mb P50 induces modest change. Mb‐facilitated diffusion has a higher poise in skeletal muscle than in myocardium. Because the basal P  O 2 hovers around 10 mmHg, Mb does not have a predominant role in facilitating O2 transport in myocardium but contributes significantly only when cellular oxygen falls below the equipoise diffusion P  O 2.

Blood flow and metabolic regulation in seal muscle during apnea

SUMMARY In order to examine myoglobin (Mb) function and metabolic responses of seal muscle during progressive ischemia and hypoxemia, Mb saturation and high-energy phosphate levels were monitored with NMR spectroscopy during sleep apnea in elephant seals (Mirounga angustirostris). Muscle blood flow (MBF) was measured with laser-Doppler flowmetry (LDF). During six, spontaneous, 8–12 min apneas of an unrestrained juvenile seal, apneic MBF decreased to 46±10% of the mean eupneic MBF. By the end of apnea, MBF reached 31±8% of the eupneic value. The t1/2 for 90% decline in apneic MBF was 1.9±1.2 min. The initial post-apneic peak in MBF occurred within 0.20±0.04 min after the start of eupnea. NMR measurements revealed that Mb desaturated rapidly from its eupenic resting level to a lower steady state value within 4 min after the onset of apnea at rates between 1.7±1.0 and 3.8±1.5% min–1, which corresponded to a muscle O2 depletion rate of 1–2.3 ml O2 kg–1 min–1. High-energy phosphate levels did not change with apnea. During the transition from apnea to eupnea, Mb resaturated to 95% of its resting level within the first minute. Despite the high Mb concentration in seal muscle, experiments detected Mb diffusing with a translational diffusion coefficient of 4.5×10–7 cm2 s–1, consistent with the value observed in rat myocardium. Equipoise PO2 analysis revealed that Mb is the predominant intracellular O2 transporter in elephant seals during eupnea and apnea.

Interaction of fatty acid with myoglobin

Upon titration with palmitate, the 1H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb solution than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (K d) of 43 μM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.

1H-NMR characterization of the human myocardium myoglobin and erythrocyte hemoglobin signais

Abstract The 1 H-NMR signal of deoxy Mb provides a unique opportunity to measure tissue oxygenation in vivo. To utilize the technique for human application, however, requires a specific spectral characterization of both human Mb and erythrocyte Hb. We report that the proximal histidyl-NH signal of human deoxy Mb resonates at 80.3 ppm at 25°C and maintains a 3.9 ppm separation with the corresponding Hb A signal throughout the physioiogical temperature range. In the particular case of the human thenar muscle, the deoxy Mb signal is clearly detectable without any interference from Hb.

'It's hollow': the function of pores within myoglobin.

SUMMARY Despite a century of research, the cellular function of myoglobin (Mb), the mechanism regulating oxygen (O2) transport in the cell and the structure–function relationship of Mb remain incompletely understood. In particular, the presence and function of pores within Mb have attracted much recent attention. These pores can bind to Xe as well as to other ligands. Indeed, recent cryogenic X-ray crystallographic studies using novel techniques have captured snapshots of carbon monoxide (CO) migrating through these pores. The observed movement of the CO molecule from the heme iron site to the internal cavities and the associated structural changes of the amino acid residues around the cavities confirm the integral role of the pores in forming a ligand migration pathway from the protein surface to the heme. These observations resolve a long-standing controversy – but how these pores affect the physiological function of Mb poses a striking question at the frontier of biology.

Observing the deoxy myoglobin and hemogobin signals from rat myocardium in situ

1H NMR proximal histidyl NδH signals of deoxy hemoglobin (Hb) and myoglobin (Mb) are distinguishable in the rat myocardium in situ. In the normoxic resting state, the blood and tissue pO2 is sufficient to saturate both Mb and Hb. No deoxy Mb or Hb signals are detected. Under 12% inspired O2, the erythrocyte Hb is partially desaturated and yields the α and β proximal histidyl NδH signals of deoxy Hb. The detection of the Hb signals clarifies the debate about the NMR visibility of erythrocyte Hb in vivo and augments the strategy to observe tissue pO2.

Imaging apolipoprotein AI in vivo

Coronary disease risk increases inversely with high‐density lipoprotein (HDL) level. The measurement of the biodistribution and clearance of HDL in vivo, however, has posed a technical challenge. This study presents an approach to the development of a lipoprotein MRI agent by linking gadolinium methanethiosulfonate (Gd[MTS‐ADO3A]) to a selective cysteine mutation in position 55 of apo AI, the major protein of HDL. The contrast agent targets both liver and kidney, the sites of HDL catabolism, whereas the standard MRI contrast agent, gadolinium‐diethylenetriaminepentaacetic acid‐bismethylamide (GdDTPA‐BMA, gadodiamide), enhances only the kidney image. Using a modified apolipoprotein AI to create an HDL contrast agent provides a new approach to investigate HDL biodistribution, metabolism and regulation in vivo. Copyright

1H NMR Approach to Observe Tissue Oxygenation with the Signals of Myoglobin

The myoglobin technique measures oxygen tension in myocytes. It relies on a quantitative measurement of the Val E11 and His F8 signals and an accurate value for the [O2]50 for Mb. Even though the Mb oxygen affinity in the cell is in question, the NMR results still reflect the degree of Mb oxygen saturation. Although magnetic resonance has established a variety of strategies to measure tissue oxygenation, the Mb approach is the most direct and will lead to a better understanding of oxygens role in regulating cellular activity.