Ethical approval was granted for the study by the research review board at the authors’ institution. Enrollment was voluntary, and all participants signed a consent form previously approved by the institution’s ethics committee for the protection of human subjects in research. A total of 37 mothers who delivered at late preterm were registered, as well as 37 mothers who delivered at term. They were non-smokers, normotensive, and had singleton gestations ranging from 34 to 42 weeks. Gestational age at entry was determined by an obstetric estimate of the last menstrual period, uterine size, and ultrasound examination.17
Late preterm mothers delivered between 34 weeks and <37 weeks and their ages ranged from 14–44 years (mean 28 years). Term mothers delivered between 37 and 42 weeks and were aged 22–45 years (mean 33 years). No medical complications were encountered during pregnancy and all deliveries occurred vaginally and were free of artificial support. The cord blood was deemed neonatal. Late preterm neonates (20 males, 17 females) born to late preterm mothers, had a mean ± SD birth weight of 2,463 ± 415 g. Term neonates (17 males, 20 females) born to term mothers, had a mean ± SD birth weight of 3,292 ± 491 g.
Maternal venous blood samples were taken during labour from the median cubital vein into evacuated lithium heparinised tubes. Samples of umbilical cord blood were collected immediately after delivery by aseptic puncture of the umbilical veins connected to the maternal placenta into evacuated lithium heparinised tubes. The specimens were centrifuged at 4° C; cord and venous plasma were separated for assay of NO. Haemolysates were prepared from the packed cells for the assays of GPx and CAT. Measurement of NO concentration was performed in a 96-well microtitre plate using reagents from Cayman (Cayman Chemical Company, Michigan, USA) and was a two-step process based on the method of Green et al
The first step is the conversion of nitrate to nitrite utilising nitrate reductase; the second is the addition of the Greiss reagents which, on reaction with nitrite, develop a deep purple azo chromophore. Absorbance is read after 10 minutes at 540 nm using a plate reader, the Thermo Labsystem Multiskan Spectrum (Thermo Electron Corporation, Zantaa, Finland). Standards, controls and samples were measured in triplicate and expressed in μM
Haemolysates were prepared from the remaining packed cells as previously described for the GPx assay, using Ransel kits.19
Ransel controls (Randox Laboratories, Crumlin, UK) were used to monitor the GPx activity.
Catalase activity in the haemolysates and controls was assayed in a 96-well microtitre plate, using catalase assay kits (Cayman Chemical Company, Michigan, USA), which utilise the peroxidative function of CAT for determination of enzyme activity. The method is based on the reaction of CAT with methanol in the presence of an optimal concentration of H2
to form formaldehyde, measured with purpald (4-amino-3-hadrazino-5- mercapto-1,2,4-triazole) as the chromogen.20
Purpald specifically forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colourless to a purple colour.19
The absorbance was read at 540 nm using a plate reader, the Thermo Labsystem Multiskan Spectrum (Thermo Fisher Scientific, Massachusetts, USA).
The imprecision study was done using the internal quality control sera supplied in the commercial kits. Analyses of twelve aliquots of the controls revealed an intra-assay coefficient of variations (%) of 2.9, 3.6 and 4.1 for NO, GPx and CAT, respectively; the inter-assay CV values (%) performed over six days were 3.6, 5.0 and 5.4 for NO, GPx and CAT, respectively.
Each data set was confirmed to have Gaussian distribution by the one-sample Kolmogorov-Smirnoff test and, additionally, by histogram plots. Then all data were presented as mean ± SD. The mean differences between more than two groups were determined by one-way analysis of variance (ANOVA) using Scheffe’s post-hoc test for a multiple comparison. Probability (P) values were two-tailed and P <0.05 was considered to be significant.