No abstract

Recently, Bolton and Bodenhausen (1, 2) have developed a new experimental 2D technique which can be useful for structure determination and for assignment of resonances. This so-called RELAY experiment transfers coherence in two steps: hrst magnetization is transferred from one nucleus to another, e.g., one proton to another, and then this transferred magnetization is relayed to a third nucleus, e.g., a carbon-13. As vicinal proton-proton couplings are often we1 resolved, magnetization can be transferred between those protons, and pairs of adjacent protonated 13C ntEclei can then be identified simply by inspection of the 2D spectrum (I), a.n&go~Iy to the more versatile but less sensitive two-dimensional MADEQLJATE exzepiment (3-5). The RELAY experiment appears to be most valuable in cases where the resonances in the proton spectrum cannot be assigned because of severe overlap.The original RELAY experiments (1,2) require a l&&p muence, each step with different phases of the rf pulses. On some spectrometers a I&step experiment is not easily implemented, and we therefore propose a simphfred version of this experiment which requires only a 4-step sequence and also improves the sensitivity by a factor of& The pulse sequence of the four-step RELAY experiment is set out in Fig. I. The phases & and $Q are cycled according to Table 1 in the four steps of the experiment. For completeness, the way the magnetization is transferred will be discussed briegy. Consider an AMX spin system in which A and M are protons with a coupling &M and X is a 13C nucleus directly coupled to proton M. The coupling JAx is med to be zero. The first 90' proton pulse simply rotates the longitudinal magnetization into the transverse plane (Fig. 2a). The 180 ' 13C pulse at the midpoint of the l1 ,interval serves to eliminate the overall effect of heteronuclear coupling at the end of the evolution period, just before the second proton 90" pulse (Fii. 2b). This 180' pulse serves a similar function as the 180" pulse in the center of the evolution period in the decoupled version of the heteronuclear shift correlation experiment (5). The second, nonselective 90" proton pulse can, for convenience, be considered as a cz+acade of two semiselective 90" proton pulses (7), one applied to the A nucleus fohowed by one applied to the M nucleus. The hypothetical 90" A pulse changes the longit%rdinal magnetization of the A transitions and therefore the longitudinal magnetk&ons of the connected M transitions. The hypothetical M pulse, applied along the x axis, rotates the longitudinal M magnetization, which originates from spin A, along the .I; 149 0022-2364/83 $3.00

An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2-7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO 2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg ‫؍‬ 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O 2͞5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO 2 Eppendorf electrode. This work illustrates that anatomically coregistered pO 2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO 2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO 2 measurements of the same animal with useful spatial (Ϸ1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.A bnormal values of pO 2 (the partial pressure of O 2 ) are linked to many pathophysiological conditions (e.g., ischemic diseases, reperfusion injury, and oxygen toxicity). Approximately one-third of human tumors evaluated for oxygen status have shown significant oxygen deficiency, and oxygen deficiency increases the tumor's resistance toward cancer treatment modalities, including radiation and chemotherapy (1, 2). Additionally, hypoxic microenvironments in tumors are known to promote processes driving malignant progression, such as angiogenesis, elimination of p53 tumor suppressor activity, genetic instability, and metastasis (3-5). Understanding of tumor hypoxia could lead to the discovery of diagnostic and prognostic markers for malignant progression, discovery of novel therapeutic targets, and the development of new constructs for gene therapy applications in human cancer. Hence, a noninvasive technique that could accurately and repetitively measure tissue oxygenation would find broad application in clinical and basic research. Unfortunately, the currently used electrochemical method (6) for in vivo oxygen measurement is an invasive technique applicable only to accessible tumors. Further, the technique is hampered by measurements of only a small part of the total tumor, which cannot be re-evaluated. Several magnetic resonance techniques (7, 8) have been developed for in vivo oximetry, including spin label oximetry (9), MRI (10), and electron paramagnetic resonance imaging (EPRI) (11,12). The blood oxygen level-dependent...

Tumors exhibit fluctuations in blood flow that influence oxygen concentrations and therapeutic resistance. To assist therapeutic planning and improve prognosis, noninvasive dynamic imaging of spatial and temporal variations in oxygen partial pressure (pO 2 ) would be useful. Here, we illustrate the use of pulsed electron paramagnetic resonance imaging (EPRI) as a novel imaging method to directly monitor fluctuations in oxygen concentrations in mouse models. A common resonator platform for both EPRI and magnetic resonance imaging (MRI) provided pO 2 maps with anatomic guidance and microvessel density. Oxygen images acquired every 3 minutes for a total of 30 minutes in two different tumor types revealed that fluctuation patterns in pO 2 are dependent on tumor size and tumor type. The magnitude of fluctuations in pO 2 in SCCVII tumors ranged between 2-to 18-fold, whereas the fluctuations in HT29 xenografts were of lower magnitude. Alternating breathing cycles with air or carbogen (95% O 2 plus 5% CO 2 ) distinguished higher and lower sensitivity regions, which responded to carbogen, corresponding to cycling hypoxia and chronic hypoxia, respectively. Immunohistochemical analysis suggests that the fluctuation in pO 2 correlated with pericyte density rather than vascular density in the tumor. This EPRI technique, combined with MRI, may offer a powerful clinical tool to noninvasively detect variable oxygenation in tumors.