Many attempts have been made to obtain fundamental information about blood diseases by estimating the life span of the red blood cell. While a number of different clinical methods have been used, most are unreliable and none is based on the direct measurement of hemoglobin synthesis rate. The primary objective of this proposal is to develop an in vivo method to enable measurement of the rate of fractional hemoglobin synthesis and red blood cell life span, in hematologic diseases. The method has been adapted from work described in the 1940's when hemoglobin was labeled with 15N- glycine in vivo and the heme was isolated for analysis. The amino acid glycine is required for the synthesis of the protoporphyrin of hemoglobin. Progress in mass spectrometry analysis and the availability of 15N labels now make such techniques feasible. Furthermore, it will be simple and more affordable than the original method described. The secondary objective is to apply this measurement to sickle cell disease, which provides a model of changed red blood cell metabolism. Intermittent oral doses of 15N- glycine, 400mg over 12 hours will label heme and globin. The heme is ultimately lost in the stool as bilirubin and therefore this label will not be recycled to heme synthesis. Blood will be collected at times zero (pre dose), 12 and 24 hours, and the heme and globin will be isolated from the red blood cell hemolysates. The intracellular free glycine pool will be measured to represent the precursor pool enrichment for heme synthesis. The glycine will be isolated with high performance liquid chromatography, from a trichloroacetic acid extract of the red blood cell. Enrichments of 15N will be measured by mass spectrometry so that fractional hemoglobin turnover rate and red blood cell survival time can be calculated. This method will simultaneously measure rates of in vivo synthesis and degradation of fetal and adult or sickle hemoglobin. The advantages of this proposed method over the clinical use of chromium 51 (51 Cr) to tag the red cells, are the accuracy and directness of the measurement. Accuracy of the 51 Cr method is affected by leakage of chromium from the red cell after the cells are removed from the subject, tagged in vitro and then reintroduced to the subject. The tagged cells may also be metabolically perturbed by this procedure. Finally, the proposed method presents no radiation hazard and enables the dynamics of red cell metabolism to be measured, as well as the estimates of red cell survival time. In addition the procedure measures whole body protein turnover using the 15N-glycine, urine collection and mass spectrometry. This can be correlated with the fractional hemoglobin metabolism, to determine how change in the metabolism of this one protein, is reflected in the whole body. This method to measure the dynamics of hemoglobin metabolism will have wide application as a tool for in vivo characterization of all conditions resulting from altered hemoglobin synthesis. It may also eventually prove useful in measuring platelet survival and turnover rates as well as other blood constituents affected by disease.
