IN-VIVO AND IN-VITRO EVIDENCE FOR NONRESTRICTED TRANSPORT OF 2',7'-BIS(2-CARBOXYETHYL)-5(6)-CARBOXYFLUORESCEIN TETRAACETOXYMETHYL ESTER AT THE BLOOD-BRAIN-BARRIER

2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl e ster (BCECF-AM), a fluorescence reagent for the measurement of intrace llular pH with a molecular weight of 809 Da, was used to test the hypo thesis that the blood-brain barrier (BBB) does not restrict the influx of substrate with a molecular weight greater than 600 Da. Using cultu red bovine brain capillary endothelial cells (BCEC), the influx rate o f BCECF-AM was found to be 151 +/- 2 mu l/min/mg protein and was extra polated to give 446 +/- 7 mu l/min/g brain as a BBB permeability surfa ce area product (PS). No significant saturation was observed for the i nitial in vitro uptake of BCECF-AM into BCEC at concentrations 0.1, 1. 0 and 5.0 mu M. The apparent activation energy of the initial uptake o f BCECF-AM was found to be 5.09 kcal/mol. These results suggest that B CECF-AM is transported into the BBB by passive diffusion. The in vivo BBB PS value was also found to be 295 +/- 48 mu l/min/g brain and 132 +/- 24 mu l/min/g brain by the in situ brain perfusion and the carotid artery injection methods, respectively. No significant efflux of BCEC F-AM from the brain was observed over a 120 sec washout period, sugges ting that BCECF-AM is immediately hydrolyzed to BCECF, a hydrophilic a nalogue, in the brain after crossing the BBB. The octanol/water partit ion coefficient of BCECF-AM was found to be 5.66 +/- 0.27. The BBB PS value of BCECF-AM was predicted to be 105 mu l/min/g brain, based on t he relationship between the BBB PS value and the value of partition co efficient divided by the square root of the molecular weight. These re sults demonstrate that BCECF-AM transport across the BBB is not restri cted despite its large molecular size.