METHYLENE BLUE UPTAKE AND THE REVERSAL OF CHEMICALLY INDUCED METHEMOGLOBINEMIAS IN HUMAN ERYTHROCYTES

¹ Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire

Methylene blue is accumulated by human erythrocytes when added to extracellular fluid. A limited and apparently nonspecific uptake of methylene blue can always be observed, but maximal uptake requires a substrate available to the pentose phosphate shunt and is, therefore, inferred to require reduced nicotinamide adenine dinucleotide phosphate (NADPH). The nonspecific uptake, however, is adequate to activate the shunt-linked methemoglobin reductase system. If nitrite is used to generate methemoglobin, maximal methylene blue uptake occurs secondarily to methemoglobin reduction. When present in erythrocytes contemporaneously, methemoglobin formation by phenylhydroxylamine (PHA) and methemoglobin reduction by methylene blue compete for the pentose shunt; therefore, net rates of methemoglobin reduction are slower than in the case of nitrite. Concentrations of PHA and unsubstituted hydroxylamine too low to generate methemoglobin still block NADPH-linked methylene blue uptake. Thus, when red cells exposed to high concentrations of PHA are washed, residual phenylhydroxylamine prevents maximal methylene blue uptake but does not affect methemoglobin reduction rates. Even when red cells are washed after exposure to hydroxylamine, however, both NADPH-linked methylene blue uptake and methemoglobin reduction by methylene blue are blocked. Thus, hydroxylamine appears to produce an irreversible lesion of the pentose shunt. Another indication of irreversible damage to the red cell is "sulfhemoglobin" accumulation. Although a concentration-dependent spectrum probably exists for these phenomena, they explain why a given methemoglobin level generated by nitrite is more effectively reduced by methylene blue than an equivalent level generated by phenylhydroxylamine. The latter, in turn, is more efficiently reduced than a comparable methemoglobinemia generated by hydroxylamine.

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