Urine neutrophil gelatinase-associated lipocalin concentration in healthy newborns during the first three postnatal days

Introduction Urine neutrophil gelatinase-associated lipocalin (uNGAL) is a biochemical marker significant for early prediction of acute kidney injury in adults. However, it has not been examined sufficiently among the infant population, particularly newborns in terms of reference values. The aim of our study was to determine the concentration of uNGAL in healthy term newborns and to determine if there was a difference in uNGAL concentration according to gender, postnatal age and birth weight. Materials and methods Our study involved 81 healthy term newborns birth (≥ 37 weeks, Apgar score ≥ 8 in the first minute after birth, CRP < 5 mg/L). Urine NGAL was measured using chemiluminescent microparticle immunoassay (CMIA) within 72 hours after birth, on Architect plus ci8200 analyser (Abbott, Chicago, USA). Data were analysed using Statistica software. Results The median concentration of uNGAL in the whole study group of healthy term newborns was 27.1 ng/mL (16.5-56.0 ng/mL) (newborn girls, 27.1 ng/mL (15.8-47.9 ng/mL); newborn boys, 27.9 ng/mL (16.5-61.0 ng/mL), P = 0.941). Median uNGAL concentration according to postnatal age expressed in days was 28.2 ng/mL (11.7-57.2 ng/mL) 1st day, 28.9 ng/mL (16.5-64.2 ng/mL) 2nd day and 23.9 ng/mL (20.2-46.6) 3rd day, P = 0.863. Regarding birth weight for newborns < 3500 g, median concentration was 25.0 ng/mL (16.5-45.4 ng/mL) and for weight ≥ 3500 g 30.6 ng/mL (16.5-64.2 ng/mL), P = 0.455. Conclusions There were no significant difference in uNGAL concentration in relation to gender, postnatal age and birth weight.

1 population. Although NGAL can be secreted by different types of cells, some research data indicate its high diagnostic sensitivity and specificity in acute kidney injury. Numerous studies emphasise its predictive, diagnostic and prognostic benefits in acute kidney injury (2)(3)(4)(5)(6)(7)(8). To date, research on urine NGAL in newborn population has included certain conditions and illnesses such as asphyxia and sepsis, which might cause acute kidney injury. In most research, reference groups consisted Mikulić V. et al. uNGAL in healthy newborns in first three days of a small number of healthy newborns (9)(10)(11)(12). There is not enough data about NGAL concentration in healthy infants, including both serum and urine NGAL, which challenges the probability of correct interpretation in different pathological conditions. Thus, the aim of the study was to determine the concentration of urine NGAL (uNGAL) in healthy term newborns, as well as to determine if there was a difference between urine NGAL concentration in relation to gender, postnatal age and birth weight. The hypothesis was that uNGAL concentration does not depend on gender, postnatal age and birth weight.

Subjects
The present study was prospective and included 81 healthy newborn (newborns girls, N = 31 and newborn boys, N = 50) born at University Hospital Mostar, Bosnia and Herzegovina, from April to October 2019. The study was approved by the Ethics Committee of the University Clinical Hospital Mostar (Ethical approving number 2272/12 at UCHM) in adherence to the Declaration of Helsinki. Written informed consent was obtained from all parents after explaining the purpose of the study. The newborns included in the study were delivered from full-term pregnancies and inclusion criteria after clinical examination were as follows: gestational age at birth ≥ 37 weeks, Apgar score ≥ 8 in the first minute after birth, C-reactive protein (CRP) < 5 mg/L, normal physical and ultrasound examination (12)(13)(14). If routine clinical examination after birth showed the presence of infection, cardiac disease, congenital anomalies and chromosomal disorder, the newborns were excluded from the study.

Methods
The present study used the leftover serum samples that had previously been analysed in routine laboratory tests (mainly bilirubin) required by a neonatologist after the post-delivery examination. The leftover serum was used to determine CRP concentration, which was the including criteria for

Results
The study comprised 81 healthy term neonates, newborn girls (subgroup F) and newborn boys (subgroup M). The respective median gestational ages were 39 (37-41) weeks in females, and 40 (37-41) weeks in males. Median values with an interquartile range along with general characteristics of healthy newborns (birth weight, birth length and Apgar score) differentiated by gender are shown in Table 1. No parameter was shown to be statistically significantly different between genders, although we observed an almost significant difference regarding birth weight (P = 0.056). Median uNGAL concentration in the whole study group was 27.1 ng/mL (16.5-56.0 ng/mL). The values did not differ between genders (P = 0.941), in relation to postnatal age (P = 0.863) nor in relation to birth weight (P = 0.455) ( Table 2).

Discussion
The study demonstrated that there was no significant difference in uNGAL concentration in relation to gender, postnatal age and birth weight, which supports the hypothesis mentioned beforehand.
There have been several studies that included uN-GAL concentration measurement in a healthy newborn population. One of these studies, done by Chen et al., included measurement of uNGAL in 38 term newborns when they were three days old. The median concentration of uNGAL was 88.1 ng/    (18).
The only research carried out on a higher number of subjects was conducted by Kamianowska et al. (19). This research included 88 term neonates with median uNGAL concentration of 16.74 ng/mL. In contrast to our study, their study proved that uN-GAL concentration is significantly higher in female newborns. The median concentration of uNGAL was 24.16 ng/mL for female neonates and 13.30 ng/mL for male neonates, P < 0.01. The uNGAL values in these studies were lower than those obtained by us (17)(18)(19). The reasons for the reported differences may result from different measurement methods, different manufacture reagents and antibodies. Study by Krzeminska et al. on 30 healthy adults demonstrated that uNGAL concentration obtained by CMIA (Abbott) method was significantly higher than ELISA (R&D Systems) (20). It is highly important to note that the concentrations in the present study were measured using a fully standardized and automated CMIA method, as opposed to most above-mentioned studies using ELISA method from different manufacturers (16)(17)(18)(19) to all previous studies, our study had the same potential limitation -we did not use uNGAL/creatinine ratio. Our rationale was that at this early stage of life creatinine excretion is not constant and therefore cannot be used to improve the variability of the results due to urine concentration. In conclusion, since it was carried out in healthy newborns, as expected, the present study showed no significant difference in concentration of uNGAL in relation to gender, postnatal age and birth weight of new-borns. Future studies should include a higher number of neonates utilizing the same standardized method in order to precisely determine NGAL reference interval for neonate population and thus contribute to the possibility of early acute kidney injury recognition in the first days of life.