Sexual Dimorphism in Whole Blood Nitrite and Susceptibility to Hypoxic Sickle Cell Vaso-Occlusive Injury in Mice.
Authors: Hsu Lewis L. , Dejam Andre , Pelletier Mildred , Cochard Audrey E. , Diwan Bhalchandra , Gladwin Mark , Schechter Alan N. , Noguchi Constance 
Published: April 2020

Abstract
Murine vasoregulation and ischemia-reperfusion injury studies indicate greater nitric oxide synthase (NOS) activity in females than in males, linked to estrogen upregulation of endothelial NOS-3 (eNOS). Plasma levels of nitrite ions have been used as an index of NOS activity in vivo. Our research on blood nitrite in humans indicates that eNOS-derived NO is stabilized as nitrite, transported by erythrocytes, and bioactivated by a nitrite reductase activity of deoxyhemoglobin (Dejam 2005 Blood 106:734–739). Transgenic mice expressing exclusively human sickle hemoglobin (sickle mice) originally from Berkeley (Pastzy, 1997) are susceptible to vaso-occlusive ischemic injury from hypoxia, and increased NO availability may protect against this ischemic injury. Hypothesis: Females will have higher whole blood nitrite levels, which will be a marker of NO bioavailability and protection against ischemic challenges such as sickle cell vaso-occlusive liver injury after hypoxia-reoxygenation.
Methods: Whole blood samples were stabilized with a ferricyanide solution as previously described (Dejam 2005), then analyzed for nitrite levels using reductive chemiluminescence. C57BL6 mice were studied at baseline, during normobaric hypoxia (10% O2), or 1 hour after injection of the NOS inhibitor L-N5-(1-iminoethyl)-ornithine (L-NIO, 30 mg/kg) vs saline. Sickle mice had hypoxia-reoxygenation challenge to induce sickle vaso-occlusive injury: 2 hours of hypoxia followed by blood sample 18 hours later for serum alanine aminotransferase (ALT) as a marker of ischemic injury.
Results: Average whole blood nitrite was 16% (SEM 7%) lower in males than in females, with much individual variation. Hypoxic exposure as short as 15 minutes lowered nitrite by 40% (SEM 7%). The magnitude of L-NIO effect was similar, lowering nitrite by 30% (SEM 9%). Female sickle mice and C57BL6 mice of both sexes showed no rise in ALT after hypoxia-reoxygenation, but male sickle mice showed 3-fold rise in ALT (p &lt; 0.05). Ovariectomized female sickle mice had elevated ALT that was no different from males.
Discussion: Murine whole blood nitrite levels show sexual dimorphism consistent with estrogen upregulation of eNOS. L-NIO inhibition of NOS was associated with a fall in nitrite level, consistent with inhibition of nitric oxide production. The remaining nitrite after NOS inhibition is likely to be controlled by the several eNOS-independent influences on nitrite, such as dietary nitrite and nitrate, and systemic inflammation. Although hypoxia was expected to upregulate eNOS production of NO, nitrite levels fell significantly with moderate hypoxia, consistent with reduction of nitrite to NO by deoxyhemoglobin. Male sickle mice and ovariectomized females are susceptible to liver injury with hypoxic-reoxygenation challenge, consistent with a role for female hormones in upregulating protective NO bioavailability. Thus, modest changes in NO bioavailability are potentially enough to protect against sickle cell vaso-occlusive injury. Future studies to correlate whole blood nitrite levels with vasoregulation and ischemic protection are feasible with this new method for experimental measurement of whole blood nitrite.