Validity of multiple-time regression analysis in measurement of tritiated and iodinated leptin crossing the blood-brain barrier: meaningful controls

Multiple-time regression analysis has been used to study the influx of radiolabeled peptides and polypeptides across the blood-brain barrier (BBB). This study used both tritiated and iodinated leptin to clarify several issues associated with these measurements. Recombinant murine leptin was radiolabeled with H-3 by derivatization or with I-125 by the iodobead method and each studied separately in mice. Intact H-3-leptin had a higher apparent influx rate from blood to brain than did intact I-125, correlating with its higher proportion of reversible association with the capillary lumen that would misleadingly appear to reflect entry. Yet the majority of H-3-leptin and I-125-leptin reached brain parenchyma. There was no significant difference in the influx rate between cerebral cortex and the subcortical regions, thus ruling out a predominant contribution of simple diffusion through the circumventricular organs or choroid plexuses outside the BBB. The influx of radiolabeled leptin, especially I-125-leptin, was decreased by excess unlabeled leptin, supporting the presence of a saturable transport system for leptin at the BBB. To identify the specificity of the transport system and determine whether it is shared by H-3-leptin and I-125-leptin, these radioactively labeled leptins were heat-denatured. Denaturation had no effect on the fast influx of H-3-leptin, but abolished the entry of I-125-leptin into brain; excess denatured leptin failed to inhibit the influx of either H-3-leptin or I-125-leptin. This indicates that the conformation of I-125-leptin is similar to that of native unlabeled leptin, so that iodination would be the better choice for investigating the interaction of leptin with the BBB. However, H-3-leptin can use the same transport system, as shown by inhibition of its influx by unlabeled leptin, whereas the derivatization procedure altered its biophysical properties such that its non-saturated influx was greatly enhanced. Finally, the rapid influx of radioactively labeled leptin contrasted greatly with that of the reference compounds Tc-99m-albumin and H-3-inulin which had no significant penetration of the BBB. Thus, with additional considerations such as stability and interactions with the vasculature, multiple-time regression analysis is sensitive and selective for study of the penetration of peptides across the BBB. (C) 2001 Elsevier Science Inc. All rights reserved.