[14C]cyanate labeling of sheep red cells: covalent binding to hemoglobin continues in vivo for a day.

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.