Umbelliferone as an Intracellular pH-sensitive fluorescent indicator and blood-brain barrier probe: instrumentation, calibration, and analysis.

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Abstract
PH measurements were made of the intracellular compartment of brain in cats and rats using 7-hydroxycoumarin (umbelliferone). Umbelliferone is a fat-soluble, pH-sensitive fluorescent indicator with both molecular and ionic fluorophors. A nomogram relating the ratio of the indicator''s fluorescence at 450 nm from 340 and 370 nm excitation to pH is created. From this nomogram, brain pH can be determined from dual-channel recording of the indicator''s calibrated 450 nm fluorescent tissue clearance curves. Umbelliferone is nontoxic, fat soluble and freely diffusible across the blood-brain barrier. Its pKa is not altered by brain tissue. Recordings can be complicated by the hemodynamic artifact; it is necessary to focus the instrument on a small avascular field. Brain pH in the cat at normocapnia under light halothane anesthesia was 6.98 and under light barbiturate anesthesia, 7.05. Brain pH varied with arterial CO2 tensions, but not with acute metabolic acidosis or alkalosis. Brain pH in the rat under moderate barbiturate anesthesia was 7.08 at normocapnia. These measurements may reflect an equivalent intracellular pH. It is postulated, from an analysis of the clearance curves of umbelliferone, that this fat-soluble indicator followed an intracellular pathway into and out of brain, namely, capillary endothelium to glial cell to neuron. The clearance rates of umbelliferone were only partially responsive to changes in PacO2 [arterial partial pressure of carbon dioxide] and cerebral blood flow (simultaneously recorded by clearance of radioactive Xe).