Meconium proteases and antiproteases as a potential source of biomarkers for the assessment of the intrauterine environment of the fetus.

BACKGROUND: A protease-antiprotease balance is required to maintain the homeostasis of the intrauterine environment in which the fetus develops. Proteases and antiproteases accumulate in meconium exclusively during intrauterine life and are excreted after birth. METHODS: Proteomic analysis was used to investigate the protein composition in pooled 50 serial meconium portions from 10 neonates. The UniProt, BRENDA and MEROPS databases were the sources of information used to classify the meconium proteases and antiproteases among 946 proteins identified in meconium. RESULTS: A total of 265 enzymatic proteins and 33 protein inhibitors were identified in the meconium. The six main enzyme groups represented in the meconium were oxidoreductases (n = 44), transferases (n = 62), hydrolases (n = 137), lyases (n = 10), isomerases (n = 7) and ligases (n = 5). Six protease families were distinguished: serine (n = 28, 41.2% of all proteases), metallo (n = 23, 33.8%), cysteine (n = 10, 14.7%), aspartic (n = 4, 5.9%), theorine (n = 2, 2.9%) and mixed (n = 1, 1.5%) proteases. CONCLUSIONS: The well-characterized meconium-based biomarker panel of proteases and their inhibitors may be a source of important information for use in diagnosing fetal disorders and predicting postnatal health and development. The differences in the composition and function between individual meconium proteases and antiproteases confirm their association with numerous metabolic processes characteristic of the intrauterine environment.

Comparison of protease and aminopeptidase activities in meconium: A pilot study.

The successive accumulation of proteases and aminopeptidases in meconium are important physiological components of the intrauterine environment in which a fetus develops. The aim of the present study was to assess the changes in the activities of these enzymes in meconium of healthy infants, and to investigate whether there were any statistically significant associations between activity of the enzymes of interest and the mode of delivery. The activities of proteases and aminopeptidases were determined in meconium portions (n=110) using the substrates BODIPY FL casein and L-leucine-7-amido-4-methylcoumarin hydrochloride, respectively. Serial meconium samples (2-5 per neonate) were collected from healthy infants born vaginally (n=14), and by a cesarean section (n=16). Protease activity (104 RFU/h) was lower in the first meconium sample compared with the final sample from the same infant (3.99±2.03 vs. 5.76±2.24, respectively, mean ± standard deviation; P=0.004). Conversely, there was no significant difference in aminopeptidase activity (103 nM/l/h) between consecutive meconium samples (P=0.702). The ratios of the first-meconium sample enzyme activity to the last-meconium sample enzyme activity were lower for proteases compared with aminopeptidases (0.76±0.48 vs. 1.35±1.04, respectively mean ± standard deviation; P=0.014), and sustained in the infants born by a cesarean section (P=0.008). Spearman's correlation coefficient analysis between the first and last meconium samples showed the correlation increased in the infants born vaginally compared with the rest of the infants (proteases, R=0.618 vs. R=0.314; aminopeptidases, R=0.688 vs. R=0.566). Aminopeptidase activity did not exhibit any notable dynamic changes during meconium accumulation in the fetal intestine. In infants born vaginally compared with those born by a cesarean section, the activity of both proteases and aminopeptidases in the first meconium sample showed an improved correlation with the activity of the final meconium sample. This may suggest that in the intrauterine environment, during accumulation of meconium in the digestive tract of the fetus, the activity and/or levels of these enzymes and the substrates they catalyze were more stable in newborns born vaginally compared with infants born by caesarean section.

Can aminopeptidase N determined in the meconium be a candidate for biomarker of fetal intrauterine environment?

AIM: To investigate possible association of aminopeptidase N/CD13 with other parameters of possible homeostatic mechanisms in meconium for potential use in identifying intrauterine environmental stress factors during fetal and perinatal life. METHODS: Aminopeptidase N/CD13 (APN/CD13), calprotectin (CAL), myeloperoxidase (MPO), ceruloplasmin (CER), lactoferrin (LF) and interleukin-8 (IL-8) were determined using ELISA kits in 115 meconium samples collected from 30 healthy full term neonates. RESULTS: Significant correlations were established between meconium APN/CD13 [µg/g] (mean ± SD, median, range: 2.88 ± 9.90, 0.94, 0.09-91.54) and MPO (r = 0.77, p = .0000), CER (r = 0.48, p = .0000), LF (r = 0.26, p = .005), IL-8 (r = 0.44, p = .00012) but no correlation of APN/CD13 vs CAL (r = 0.15, p > .05). With increased APN/CD13 concentrations there were increases (p < .05) in concentrations of MPO, CER, LF and L-8. CONCLUSIONS: Meconium APN/CD13 demonstrates characteristic associations with other proteins involved in the regulation of metabolic processes. The panel of APN/CD13, MPO, CER and LF may be candidate biomarker for disorders developing in utero which may have impact on health in later life.

Meconium microbiome as a new source of information about long-term health and disease: questions and answers.

OBJECTIVE: The objective of this study is to assess the diagnostic role of meconium microbiota as a source of information about the intrauterine environment of the developing fetus and possibly health and disease in later life. METHODS: The literature review of over 30 papers published in international journals in the years 2001-2017, on the bacterial composition of meconium and early feces, investigated by metagenomic DNA sequencing in experimental studies on animals and clinical studies in neonates born after normal and pathological pregnancies. RESULTS: The bacterial composition of meconium reflects the in utero microbial environment. Bacterial colonization of the fetal gut is a source of microbial stimulation and may provide a primary signal for the maturation of a balanced postnatal innate and adaptive immune system. Clarification of a possible relationship between the presence of specific bacteria in meconium and their active role in the abnormal course of pregnancy may improve our knowledge of the pathomechanisms modifying the intrauterine environment with short- and long-term effects on the immune system and metabolic pathways. CONCLUSION: Diversified intrauterine microbiome may modify the environment of the developing fetus with possible short- and long-term impact on the individual's health and disease. Meconium which provides the individual-specific information about the intrauterine microbiome composition is a biological material with potential uses in routine clinical diagnostic practice.

Correlation between the concentrations of lactoferrin and neutrophil gelatinase-associated lipocalin in meconium.

Neutrophil gelatinase-associated lipocalin (NGAL) and lactoferrin (Lf) are among the key components of the innate immune system due to their ability to bind iron with high affinity and thus control inflammation. The aim of this study was to test the use of NGAL and LF measurements in meconium for the assessment of the intrauterine homeostasis. NGAL and Lf concentrations were measured using ELISA kits in all serial meconium portions (n = 81) collected from 20 healthy neonates. Mean ± SD meconium concentration of Lf was 45.07 ± 78.53 µg/g and more than 1000-fold higher compared with that of NGAL at 1.93 ± 2.46 ng/g. The correlation between the two proteins (r = 0.83, p < 0.0001) was found only for portions with Lf concentrations > 25 µg/g. High variability of NGAL and Lf concentrations in meconium and their correlations prove their key role as biomarkers of the fetal condition in utero. NGAL and Lf measured in meconium are candidate biomarkers for fetal iron status.

Total proteolytic activity and concentration of alpha-1 antitrypsin in meconium for assessment of the protease/antiprotease balance.

BACKGROUND: During intrauterine life, various proteolytic enzymes and their main inhibitor, alpha-1 antitrypsin, accumulate naturally in meconium. A protease/antiprotease balance is required to maintain the biological stability of the environment in which the fetus develops. METHODS: The pool of active proteases was determined using the EnzChek Protease Assay Kit. The concentration of alpha-1 antitrypsin in meconium was measured by enzyme-linked immunosorbent assay. Serial portions of meconium (n=80) were collected from healthy full-term neonates (n=19). RESULTS: Mean concentrations of active proteases and alpha-1 antitrypsin were 1.55 [standard deviation (SD) 1.3]mgg-1 (range 0.15-6.17) and 3.72 (SD 1.78)mgg-1 (range 0.76-8.55), respectively, with significant correlation (Rs=0.32, p=0.004). A significant increase in the concentration of active proteases was found between the first and last meconium portions (p<0.05). The proteases in the last meconium portions had a higher reaction velocity and affinity for the substrate than the proteases in the first meconium portions. The active protease:alpha-1 antitrypsin ratio was <0.5 in all first meconium portions, but was higher in the last meconium portions. CONCLUSIONS: Strong correlation between the concentrations of active proteases and alpha-1 antitrypsin in meconium may indicate their mutual interaction in the intrauterine environment. Alpha-1 antitrypsin maintains the protease/antiprotease balance during fetal development.