A critical evaluation of the brain efflux index method as applied to the nitric oxide synthase inhibitor, aminoguanidine.

The Brain Efflux Index (BEI) method is an in vivo procedure designed to quantitate saturable efflux mechanisms resident at the blood--brain barrier (BBB). The present work utilized the BEI method to assess the BBB efflux mechanisms of [(14)C]aminoguanidine, a nitric oxide synthase inhibitor. The BEI for [(14)C]aminoguanidine was >100% (relative to [(3)H]inulin diffusion) over a range of 41-184 pmol after 40 min. The unusually high retention (>100%) of [(14)C]aminoguanidine suggested brain parenchymal sequestration, either by neuronal uptake or tissue protein binding. The uptake of [(14)C]aminoguanidine in dendritic neuronal endings (synaptosomes) showed a saturable concentration dependency, consistent with a carrier-mediated process. Nonlinear least-squares regression yielded the following Michaelis--Menten and diffusional (k(ns)) parameters for synaptosomal [(14)C]aminoguanidine uptake: V(max)=118.50 +/- 28.77 pmol x mg protein(-1)/3 min; K(m)=58.34 +/- 8.33 muM; k(ns)=0.15 +/- 0.029 pmol x mg protein(-1)/3 min/muM; mean +/- SEM; n=3 concentration profiles). Protein binding studies using brain tissue showed negligible binding. In summary, this work identified three principle findings: (1) An apparent lack of quantifiable aminoguanidine BBB efflux; (2) a previously undescribed synaptosomal accumulation process for aminoguanidine; and (3) an interesting limitation of the BEI technique where unusual brain parenchymal sequestration yields values >100%.