ABSTRACT. Phenylhydrazine (PHZ) exposure is used to study in vitro red cell aging mechanisms dependent on Hb oxidation. The effect of PHZ on normal neonatal red blood cells was studied in unseparated blood and after separation into light and heavy cells. PHZ Neonatal RBC differ from adult RBC in several of their characteristics. Most of the intracellular Hb is HbF, a highaffinity Hb adapted to intrauterine life (1). Most of the enzymatic activities in neonatal RBC are greater than those in adult RBC and may be attributed to their younger mean age (2). However, it is known that neonatal RBC have a shorter life-span (2, 3). The cause of this shortened survival time has not yet been clarified. Recently, red cell senescence in adults has been correlated with the oxidation of intracellular Hb to methemoglobin and hemichromes (4). Low et al. (4) have shown that exposure of normal cells to PHZ oxidizes the Hb to methemoglobin and hemichromes. These oxidized Hb products attach themselves to the intracellular portion of band 3 membrane protein, causing changes in surface antigenicity and increased IgG binding. Similar changes have been demonstrated in untreated cells containing pathologic Hb, such as Hb Koln, known for its high affinity to oxygen and its lack of stability (5).Our purpose was to assess the impact of oxidative stress, induced by low dose of PHZ, on neonatal erythrocytes, specifically: 1 ) to study Hb oxidation in neonatal and adult cells by two methods (measuring total oxidized Hb by spectrophotometry and identifying specific oxidation products by Mossbauer spectra) and 2) to see whether neonatal heavy cells, enriched with old cells, are more sensitive to oxidation than lighter and younger cells.
MATERIALS AND METHODS
Collection and preparation of blood samples. Blood sampleswere obtained from umbilical veins immediately after delivery. Newborn infants, whose mothers had experienced normal pregnancies, were all healthy and mature. Blood counts and glucose-6-phosphate dehydrogenase activity were normal. Control studies were performed using blood drawn from healthy adult volunteers according to the conditions of the Helsinki Declaration. The blood was collected into heparinized tubes; the buffy coat and plasma were removed and the cells were washed three times in PBS, pH 7.4.The cells were studied both as a whole, unsevarated vovulation and after separation on a Percoll gradient. he cellskere separated into 10% high ("old") and 10% low ("young") density cells, according to Ellory and Young (6). The cells were exposed to PHZ at final concentrations of 0.1-10 mM in PBS at hematocrit of 20%, pH 7.4, for 45 min at 37°C (7). Parallel control studies were performed by means of incubation of cells in PBS alone at 37°C or room temperature.