Measurement of hemoglobin synthesis rate in vivo using a stable isotope method

We developed a method to measure hemoglobin synthesis rate (SynHb) in humans, assuming that free glycine in the red blood cell (RBC) represents free glycine in bone marrow for hemoglobin synthesis. The present rat study examines this assumption of the method and quantifies SynHb in rats. Sprague-Dawley rats (n = 9) were studied, [2-¹³C]glycine was intravenously infused over 24 h (2.5 mg kg^-1 h^-1), blood was drawn for glycine and heme isolation, and bone marrow was harvested for glycine isolation. Isotopic enrichments of glycine and heme were measured, fractional hemoglobin synthesis rate (fSynHb% day^-1) was calculated, and from this a value for SynHb (mg g^-1 day^-1) was derived. Mean body weight was 446 ± 10 g (mean ± SE) and hemoglobin concentration was 14 ± 0.5 g dl^-1. At 24 h, the mean isotopic enrichment, atom percentage excess (APE), of the RBC free glycine (1.56 ± 0.18 APE) was similar to the bone marrow (1.68 ± 0.15 APE). The rate of incorporation of ¹³C into heme increased over time from 0.0004 APE/h between 6 and 12 h, to 0.0014 APE/h between 12 and 18 h, and 0.0024 APE/h between 18 and 24 h. Consequently, fSynHb (1.19 ± 0.32, 2.92 ± 0.66, and 4.22 ± 0.56% day^-1, respectively) and SynHb (0.11 ± 0.03, 0.28 ± 0.05, and 0.42 ± 0.05 mg g^-1 day^-1, respectively) showed similar patterns over the 24-h study period. We conclude that (1) enrichment of free glycine in the circulating RBC approximates enrichment of bone marrow free glycine for heme formation and (2) this pattern of hemoglobin synthesis rate is reflecting the characteristic release and gradual maturation of reticulocytes in the circulation.