Kinetic Analysis of Cerebrovascular Isoleucine Transport from Saline and Plasma

The concentration dependence of regional isoleucine transport across the blood–brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [ Am. J. Physiol. 247 , H484–493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis‐Menten saturation kinetics where V max = 9–11 × 10 −4 μmol · s −1 · g −1 and K m = 0.054–0.068 μmol · ml −1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to · 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent K m for isoleucine transport from plasma by ≥24‐fold to 1.5–1.7 μmol · ml −1 . These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood–brain barrier. In addition, they indicate that the cerebrovascular transfer‐site affinity (1/ K m ) for isoleucine is ∼fivefold greater than previously reported with the brain uptake index technique.