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

ABSTRACT: 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.

SUBMITTER: Skarzynska E

PROVIDER: S-EPMC7323450 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Skarżyńska Ewa E Wilczyńska Paulina P Kiersztyn Bartosz B Żytyńska-Daniluk Joanna J Jakimiuk Artur A Lisowska-Myjak Barbara B

Biomedical reports 20200609 2

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 det ...[more]

PMID: 32607236

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Project description:Background: Hypospadias is a male congenital malformation that occurs in ~2 of 1,000 births. The association between hypospadias and fetal exposure to environmental chemicals has been studied, but the results are inconsistent. Although several petroleum and chlorinated solvents are suspected to have teratogenic effects, their role in the occurrence of hypospadias has been little studied and never using biomarkers of exposure. We aimed to evaluate the association between fetal exposure to petroleum and chlorinated solvents measured in meconium and the occurrence of hypospadias. Methods: We conducted a pilot case-control study in the maternity of the University Hospital of Rennes (France). Eleven cases of hypospadias and 46 controls were recruited between October 2012 and January 2014. Data from hospital records and maternal self-reported questionnaires, including socio-demographic characteristics and occupational and non-occupational exposure to chemicals, were collected. Meconium samples were collected using a standardized protocol. Levels of petroleum solvents (toluene, benzene, ethylbenzene, and p, m, and o xylene), certain metabolites (mandelic acid, hippuric acid, methylhippuric acid, S-phenylmercapturic acid, S-benzylmercapturic acid, and phenylglyoxylic acid), and two chlorinated solvents (trichloroethylene and tetrachloroethylene) were measured in meconium by gas and liquid chromatography, both coupled to tandem mass spectrometry. Associations between the concentration of each chemical and the occurrence of hypospadias were analyzed using exact logistic regressions adjusted for maternal age, educational level, pre-pregnancy body mass index, and alcohol, and tobacco consumption during pregnancy. Results are presented with odds ratios (ORs) and their 95% confidence intervals (CIs). Results: Quantification rates for petroleum and chlorinated solvents or metabolites ranged from 2.2% (for methylhippuric acid) to 77.1% (for trichloroethylene) of the meconium samples. We found a significant association between the quantification of phenylglyoxylic acid (metabolite of styrene and ethylbenzene) in the meconium and a higher risk of hypospadias (OR = 14.2, 95% CI [2.5-138.7]). The risk of hypospadias was non-significantly elevated for most of the other solvents and metabolites. Conclusion: This exploratory study, on a limited number of cases, suggests an association between petroleum solvents and hypospadias. Additional studies are needed to confirm these results and identify the determinants for the presence of these solvents in meconium.

Project description:The establishment of human gut microbiota commences initially in utero. Meconium-the first fecal material passed after birth-can be used to study fetal gut contents; however, processing meconium samples for microbiome studies presents significant technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture and contains high concentrations of PCR inhibitors. This study aimed to evaluate four different DNA extraction methods to elucidate the most effective method for bacterial DNA recovery and sequencing analysis from first-pass meconium. Samples from five infants were collected and processed using the following extraction kits: (1) Qiagen QIAamp DNA Stool Mini (QS); (2) Qiagen QIAamp DNA Microbiome (QM); (3) MoBio PowerSoil (PS); (4) MoBio MagAttract PowerMicrobiome (PM). Additionally, Kit PM was employed with a double inhibitor removal treatment (IRT) step (PM2). Bacterial DNA recovery was assessed by qPCR. Any PCR inhibition in samples was measured by spiking DNA eluates with 0.1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR quantitation. Kit PM yielded the highest average total DNA yield (79.3 ng per gram of meconium). Samples extracted with kit PS had the highest detectable levels of 16S rRNA gene by qPCR. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QS, QM, PS, and PM recovering 87.1, 91.0, 88.8, and 37.9% GBS DNA, respectively. Double IRT improved the performance of kit PM, increasing GBS recovery to 56.5%. However, once DNA yield was normalized to the level recovered with the other kits 100% of GBS DNA was detected, suggesting that levels of PCR inhibitors are related to DNA yield from kit PM. Ion Torrent 16S rRNA gene sequencing revealed a high level of inter-kit variation in meconium microbiome structure. In particular, kit QM showed a bias toward extracting Firmicute DNA, while the other kits extracted primarily Proteobacterial DNA. Choice of extraction kit greatly impacts on the ability to extract and detect bacterial DNA in meconium and on the microbiome community structure generated from these samples.

Dataset Information

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

Publications

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

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