Kris T. Huang

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 μmol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control

Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer--the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40-60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation.

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.

Volumetric Spectroscopic Imaging of Glioblastoma Multiforme Radiation Treatment Volumes

PURPOSE Magnetic resonance (MR) imaging and computed tomography (CT) are used almost exclusively in radiation therapy planning of glioblastoma multiforme (GBM), despite their well-recognized limitations. MR spectroscopic imaging (MRSI) can identify biochemical patterns associated with normal brain and tumor, predominantly by observation of choline (Cho) and N-acetylaspartate (NAA) distributions. In this study, volumetric 3-dimensional MRSI was used to map these compounds over a wide region of the brain and to evaluate metabolite-defined treatment targets (metabolic tumor volumes [MTV]). METHODS AND MATERIALS Volumetric MRSI with effective voxel size of ∼1.0 mL and standard clinical MR images were obtained from 19 GBM patients. Gross tumor volumes and edema were manually outlined, and clinical target volumes (CTVs) receiving 46 and 60 Gy were defined (CTV46 and CTV60, respectively). MTVCho and MTVNAA were constructed based on volumes with high Cho and low NAA relative to values estimated from normal-appearing tissue. RESULTS The MRSI coverage of the brain was between 70% and 76%. The MTVNAA were almost entirely contained within the edema, and the correlation between the 2 volumes was significant (r=0.68, P=.001). In contrast, a considerable fraction of MTVCho was outside of the edema (median, 33%) and for some patients it was also outside of the CTV46 and CTV60. These untreated volumes were greater than 10% for 7 patients (37%) in the study, and on average more than one-third (34.3%) of the MTVCho for these patients were outside of CTV60. CONCLUSIONS This study demonstrates the potential usefulness of whole-brain MRSI for radiation therapy planning of GBM and revealed that areas of metabolically active tumor are not covered by standard RT volumes. The described integration of MTV into the RT system will pave the way to future clinical trials investigating outcomes in patients treated based on metabolic information.

Post-radiotherapy prostate biopsies reveal heightened apex positivity relative to other prostate regions sampled

BACKGROUND AND PURPOSE Prostate biopsy positivity after radiotherapy (RT) is a significant determinant of eventual biochemical failure. We mapped pre- and post-treatment tumor locations to determine if residual disease is location-dependent. MATERIALS AND METHODS There were 303 patients treated on a randomized hypofractionation trial. Of these, 125 underwent prostate biopsy 2-years post-RT. Biopsy cores were mapped to a sextant template, and 86 patients with both pre-/post-treatment systematic sextant biopsies were analyzed. RESULTS The pretreatment distribution of positive biopsy cores was not significantly related to prostate region (base, mid, apex; p=0.723). Whereas all regions post-RT had reduced positive biopsies, the base was reduced to the greatest degree and the apex the least (p=0.045). In 38 patients who had a positive post-treatment biopsy, there was change in the rate of apical positivity before and after treatment (76 vs. 71%; p=0.774), while significant reductions were seen in the mid and base. CONCLUSION In our experience, persistence of prostate tumor cells after RT increases going from the base to apex. MRI was used in planning and image guidance was performed daily during treatment, so geographic miss of the apex is unlikely. Nonetheless, the pattern observed suggests that attention to apex dosimetry is a priority.