Increased activity of the respiratory burst in cord blood neutrophils: Kinetics of the NADPH oxidase enzyme system in subcellular fractions

Previous studies with neutrophils from new-bom infants compared to neutrophils from healthy adults have documented increased respiratory burst activity including enhanced superoxide anion (02~) production, nitro-blue tetrazoleum dye reduction, and hexose monophosphate shunt activity. To investigate the biochemical basis for these observations, we examined oxidative metabolism in membrane-rich fractions of neutrophils. Neutrophils from cord blood of vaginally delivered term infants or healthy adults were disrupted by nitrogen cavitation and subcellular fractions collected on discontinuous sucrose density gradients. Subcellular fractions of newborn neutrophils separated in a fashion identical with samples from healthy adults. Activity of alkaline phosphatase, a plasma membrane marker, was increased 4- to 5-fold in disrupted cells free from nuclei (postnuclear supernatant) as well as plasma membrane fractions from newborn samples compared to those from healthy adults. Content of lactoferrin, a specific granule marker, was decreased in postnuclear supernatants but equivalent in specific granule fractions of newborn cells compared to those from adults. No differences were noted in myeloperoxidase content of postnuclear supernatants or any other subcellular fraction. Plasma membrane fractions from phorbol myristate acetate-stimulated cord blood neutrophils made significantly more 02~ than samples from adults (newborn 32.9 ± 8.1 nmol 02~/ min/mg protein mean ± SEM, n = 3 versus adult 10.8 ± 4.2, n = 3; p < 0.05). Plasma membrane-rich fractions were also collected by the technique of differential centrif-ugation and kinetic parameters of the NADPH-dependent oxidase enzyme(s) were measured for vaginally delivered newborn and adult samples. The Kmapp for NADPH for newborn fractions was significantly increased compared with adult samples (newborn 66 ± 10 /xM, n = 6 versus adult 30 ± 6, n = 6; p < 0.025) but not to the extent that would be associated with abnormal cell function. In contrast, Vmax of newborn membrane-rich fractions was 1.7 times that of adult samples (newborn 30.6 ± 27 nmol 02-/ min/mg protein, n = 6 versus adult 18.0 ± 4.0, n = 6; p < 0.025). Plasma membrane-rich fractions from term infants delivered by cesarean section without labor had a Kmapp of 67 ± 20 tiM, « = 5 which was different from adult samples (p < 0.05) and a Vmax for 02" production of 14.8 ± 4.5 which was less than that measured from samples from vaginally delivered infants (p < 0.01). The Kmapp results imply a qualitative difference in the fetal oxidase enzyme system. In addition, the increased 02~ production and Vmax demonstrated in samples from vaginally delivered infants as compared to infants delivered by cesarean section or healthy adults suggests an effect of parturition on the fetal neutrophil which is similar to the effect of certain compounds such as lipopolysaccharide. The increased catalytic activity of the NADPH oxidase enzyme system in neutrophils from newborns may reflect "priming" during parturition.