Mock D M, Strauss R G, Lankford G L
Department of Pediatrics University of Arkansas for Medical Sciences Arkansas Children's Hospital, Little Rock 72202, USA.
Pediatr Res. 1997 Mar;41(3):424-9. doi: 10.1203/00006450-199703000-00020.
The sheep is a useful model to study fetal and newborn physiology including perinatal erythropoiesis and red cell kinetics. A practical, economical method for measuring red cell survival (RCS) in sheep would be very valuable. However, 51Cr is unsatisfactory, and suitable alternatives have not been published. In the course of investigating [14C]cyanate as a label for sheep red cells, we observed continued covalent labeling over 24 h in vivo that was great enough to introduce a substantial artifact into two commonly used parameters of RCS: posttransfusion recovery (PTR24) and time to 50% decrease (T50) when referenced to time zero. In a simulation of in vivo conditions, the amount of 14C bound to Hb increased 26 +/- 6% (mean +/- 1 SD, n = 11) over 24 h. To investigate the mechanism of the increasing 14C bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separation were used separately or in combination to quantitate intracellular free 14C and 14C covalently bound to intracellular proteins. Free 14C decreased as protein-bound [14C]cyanate increased. These studies provide evidence that covalent binding of [14C]cyanate to intracellular Hb continues in vivo for the first 24 h and that the source of the increase is intracellular free [14C]cyanate. We conclude that 1) PTR24 cannot be accurately determined by [14C]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and 2) RCS by [14C]cyanate should be referenced to blood concentrations at 24 h.
绵羊是研究胎儿和新生儿生理学（包括围产期红细胞生成和红细胞动力学）的有用模型。一种实用、经济的测量绵羊红细胞存活（RCS）的方法将非常有价值。然而，51Cr并不理想，且尚未发表合适的替代方法。在研究[14C]氰酸盐作为绵羊红细胞标记物的过程中，我们观察到在体内24小时内持续存在共价标记，其程度足以在RCS的两个常用参数中引入大量假象：输血后恢复率（PTR24）和相对于零时的50%下降时间（T50）。在体内条件模拟中，与血红蛋白结合的14C量在24小时内增加了26±6%（平均值±1标准差，n = 11）。为了研究14C结合增加的机制，分别或联合使用酸-丙酮萃取、分子筛色谱和密度梯度分离来定量细胞内游离14C和与细胞内蛋白质共价结合的14C。随着与蛋白质结合的[14C]氰酸盐增加，游离14C减少。这些研究提供了证据，表明[14C]氰酸盐与细胞内血红蛋白的共价结合在体内最初24小时内持续存在，且增加的来源是细胞内游离[14C]氰酸盐。我们得出结论：1）除非在输注前孵育标记红细胞以使氰酸盐反应接近完成，否则[14C]氰酸盐无法准确测定PTR24；2）[14C]氰酸盐的RCS应参考24小时时的血液浓度。
