Effects of Radiochemical Impurities on Measurements of Transfer Constants for [C]Sucrose Permeation of Normal and Injured Blood–Brain Barrier of Rats

Radiolabeled sucrose is often used to assess blood–brain barrier (BBB) injury in the rat, but published transfer constants (K_is) for sucrose permeation of the intact BBB (control K_is) are highly discrepant. A potential problem with the commonly used tracer, ¹⁴C(U)]sucrose, is radiolytic generation, preuse, of radiocontaminants that might readily penetrate the BBB. How such contaminants might affect measurements of sucrose K_is was examined for both the intact and the ischemically injured BBB. Three stocks of ¹⁴C(U)]sucrose were studied: newly purchased (“new”), 4-year-old, and 7-year-old. A high purity (99.9%) “new” and a 2-year-old stock of ³H(fructose-1)]sucrose were also tested. Pentobarbital-anesthetized male Sprague–Dawley rats were injected IV with each tracer separately (six to eight rats) and K_is in five brain regions were measured by the multiple-time graphical method. The “new” ¹⁴C-, “new” ³H-, and 2-year-old ³H-sucrose yielded comparable K_is, ranging from 1.2 ± 0.1 to 2.4 ± 0.3 nl · g⁻¹ · s⁻¹ (mean ± SE) across the regions. The two old stocks of ¹⁴C-sucrose yielded significantly higher regional K_is: 5.1–6.3 (4-year-old) and 8.4–9.7 (7-year-old). Thin-layer chromatography of the three ¹⁴C-tracers revealed that each contained radioimpurities (ca. 2% in both the “new” and 4-year-old, and 9% in the 7-year-old), but that the old stocks contained larger amounts of relatively mobile (more lipophilic) impurities, which can be suspected as the main cause of the elevated K_is obtained. Additional rats were subjected to 10 min of cerebral ischemia, which effects a delayed BBB injury, and 6 h later the “new” ³H- and the 4-year-old ¹⁴C-sucrose were injected together. The K_is for both tracers were elevated by like, absolute amounts (ΔK_is), but by very different percentages, over their disparate baseline values in uninjured rats (for striatum and hippocampus, the most injured regions, ΔK_is were 3.9 to 4.4 nl · g⁻¹ · s⁻¹). It is concluded that radiolysis of ¹⁴C(U)]sucrose yields certain labeled products that readily cross the BBB and that can seriously distort baseline K_is, even if present only in very small amounts. While this appears not to compromise assessment of BBB injury, definition of the authentic range of baseline, sucrose K_is for the rat BBB would appear to remain a challenge.