[C-14] 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 incl uding perinatal erythropoiesis and red cell kinetics. A practical, eco nomical method for measuring red cell survival (RCS) in sheep would be very valuable. However, Cr-51 is unsatisfactory, and suitable alterna tives have not been published. In the course of investigating [C-14]cy anate as a label for sheep red cells, we observed continued covalent l abeling over 24 h in vivo that was great enough to introduce a substan tial artifact into two commonly used parameters of RCS: posttransfusio n recovery (PTR(24)) and time to 50% decrease (T-50) when referenced t o time zero. In a simulation of in vivo conditions, the amount of C-14 bound to Hb increased 26 +/- 6% (mean +/- 1 SD, n = 11) over 24 h. To investigate the mechanism of the increasing C-14 bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separ ation were used separately or in combination to quantitate intracellul ar free C-14 and C-14 covalently bound to intracellular proteins. Free C-14 decreased as protein-bound [C-14]cyanate increased. These studie s provide evidence that covalent binding of [C-14]cyanate to intracell ular Hb continues in vivo for the first 24 h and that the source of th e increase is intracellular free [C-14]cyanate. We conclude that I) PT R(24) cannot be accurately determined by [C-14]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and 2) RCS by [C-14]cyanate should be reference d to blood concentrations at 24 h.