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A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep

¹Center for Perinatal Biology and ²Department of Obstetrics and Gynecology, Division of Perinatal Medicine, and ³Department of Pediatrics, Division of Neonatology, School of Medicine, Loma Linda University, Loma Linda, California; and ⁴Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts

Submitted 24 April 2007 ; accepted in final form 3 July 2007

The reaction of nitrite with deoxyhemoglobin results in the production of nitric oxide and methemoglobin, a reaction recently proposed as an important oxygen-sensitive source of vasoactive nitric oxide during hypoxic and anoxic stress, with several animal studies suggesting that nitrite may have therapeutic potential. Accumulation of toxic levels of methemoglobin is suppressed by reductase enzymes present within the erythrocyte. Using a novel method of measuring methemoglobin reductase activity in intact erythrocytes, we compared fetal and adult sheep and human blood. After nitrite-induced production of 20% methemoglobin, the blood was equilibrated with carbon monoxide, which effectively stopped further production. Methemoglobin disappearance was first order in nature with specific rate constants (k x 1,000) of 12.9 ± 1.3 min^–1 for fetal sheep, 5.88 ± 0.26 min^–1 for adult sheep, 4.27 ± 0.34 for adult humans, and 3.30 ± 0.15 for newborn cord blood, all statistically different from one another. The effects of oxygen tensions, pH, hemolysis, and methylene blue are reported. Studies of temperature dependence indicated an activation energy of 8,620 ± 1,060 calories/mol (2.06 kJ/mol), appreciably higher than would be characteristic of processes limited by passive membrane diffusion. In conclusion, the novel methodology permits absolute quantification of the reduction of nitrite-induced methemoglobin in whole blood.

methemoglobin reductase; fetus; whole blood