Meconium Transferrin and Ferritin as Markers of Homeostasis in the Developing Fetus.

The molecular mechanisms regulating homeostasis in the developing fetus have not been satisfactorily elucidated. Meconium contains substances accumulated in the fetal intestines. Measurements of transferrin and ferritin concentrations in meconium and assessment of transferrin-ferritin relationships could enhance knowledge about specific processes of the intrauterine period involving the two proteins and their effects on the development and growth of the fetus. Transferrin and ferritin concentrations were measured by ELISA in the homogenates of first meconium portions from 125 neonates. Higher birth weight was associated with lower ferritin concentrations in meconium (r = -0.22, p = 0.015). In neonates with a birth weight of more than 3750 g, there was a positive correlation between transferrin and ferritin concentrations (r = 0.51, p = 0.003). With meconium transferrin concentrations above 43.52 µg/g, a negative correlation between transferrin and ferritin was established (r = -0.37, p = 0.036), while with transferrin concentrations below 43.52 µg/g, the correlations between the birth weight and the meconium transferrin and ferritin concentrations were negative (r = -0.61, p < 0.001 and r = -0.43, p = 0.017, respectively). Measurements of transferrin and ferritin in meconium specimens create a new use for these common biomarkers to improve our understanding of the effects of homeostasis in utero on the fetal development and growth. Establishing reference ranges of meconium transferrin and ferritin concentrations and their association with the clinical parameters during pregnancy could aid in the assessment of the impact of intrauterine life on the health status of the neonate and its adaptation to extrauterine life.

Investigation of the Changes in Concentrations of Vitamin D-Binding Protein and Lactoferin in Plasma and Peritoneal Fluid of Patients with Endometriosis.

An evaluation of the association between the concentrations of vitamin D-binding protein and lactoferrin in the plasma and peritoneal fluid may facilitate the elucidation of molecular mechanisms in endometriosis. Vitamin D-binding protein and lactoferrin concentrations were measured by ELISA in plasma and peritoneal fluid samples from 95 women with suspected endometriosis as classified by laparoscopy into groups with (n = 59) and without endometriosis (n = 36). There were no differences (p > 0.05) in the plasma and peritoneal fluid concentrations of vitamin D-binding protein and lactoferrin between women with and without endometriosis. In women with endometriosis, there was a significant correlation between plasma and peritoneal fluid vitamin D-binding protein concentrations (r = 0.821; p = 0.000), but there was no correlation between lactoferrin concentrations in those compartments (r = 0.049; p > 0.05). Furthermore, in endometriosis, lactoferrin was found to correlate poorly with vitamin D-binding protein (r= -0.236; p > 0.05) in plasma, while in the peritoneal fluid, the correlation between those proteins was significant (r = 0.399; p = 0.002). The characteristic properties of vitamin D-binding protein and lactoferrin and the associations between their plasma and peritoneal fluid concentrations found in women with endometriosis may provide a novel panel of markers to identify high-risk patients in need of further diagnostic measures.

Links Between Vitamin D-Binding Protein, Alpha-1 Antitrypsin and Neutrophil Proteins in Meconium.

BACKGROUND/AIMS: Alpha-1 antitrypsin (AAT), vitamin-D binding protein (VDBP) and neutrophil granule proteins are specifically related to the neutrophil function and may be considered candidate biomarkers detected and measured in meconium (the first feces of newborn infants) as signals indicating abnormal processes in the fetal stage. Individual proteins found in meconium can be a source of information pertaining to the intrauterine metabolic processes. METHODS: Concentrations of AAT, VDBP, calprotectin, myeloperoxidase, lactoferrin and elastase were measured using ELISA tests in 80 meconium samples collected from 19 healthy, full-term neonates. RESULTS: The meconium concentrations of VDBP and AAT (mean±SD, [mg/g meconium]: 3.74±6.93, 3.72±1.79, respectively) were approximately 1000 times higher than those of the protein granule proteins calprotectin, myeloperoxidase, elastase and lactoferrin (mean ± SD, [µg/g meconium]: 285.7±215.8, 1.83±1.73, 1.72±2.70, 45.58±78.89, respectively). The correlation between VDBP and AAT was negative (r= - 0.40. p=0.000) and those between VDBP and calprotectin (r=0.38, p=0.000) and VDBP and myeloperoxidase (r=0.45, p=0.000) were positive. AAT was found to correlate positively with lactoferrin (r=0.38, p=0.000). CONCLUSION: The correlations between the concentrations of VDBP and AAT, and with neutrophil granule proteins observed in meconium indicate their functional relationship in the intrauterine environment of the developing fetus. Meconium can be seen as an apparently underutilized source of biomarkers for evaluation of metabolic processes specific to fetal development.

The Influence of Lactoferrin in Plasma and Peritoneal Fluid on Iron Metabolism in Women with Endometriosis.

The aim of this study was to investigate the relationship between lactoferrin and iron and its binding proteins in women with endometriosis by simultaneously measuring these parameters in plasma and peritoneal fluid. Ninety women were evaluated, of whom 57 were confirmed as having endometriosis. Lactoferrin was measured by ELISA, transferrin, ferritin and iron on a Cobas 8000 analyser. Lactoferrin and transferrin in peritoneal fluid were lower compared to plasma, in contrast to ferritin and iron. In plasma, lactoferrin showeds associations with iron and transferrin in endometriosis and with ferritin in the group without endometriosis. Lactoferrin in peritoneal fluid correlated with lactoferrin, iron and transferrin of plasma in patients without endometriosis. The ratio of lactoferrin concentration in peritoneal fluid to plasma differentiated stage I versus IV of endometriosis and was negatively correlated with the iron ratio in patients without endometriosis. The ferritin ratio differentiated women with and without endometriosis. The very high ferritin ratios, especially in advanced stages of endometriosis, suggest the protective involvement of this protein in peritoneal fluid and the loss of this role by lactoferrin. The results demonstrate the validity of assessing iron metabolism in women with endometriosis, which may be useful as a marker of the disease and its progression.

Exploring associations of anthropometric parameters and serum triglycerides with serum thyroid hormones in young women.

Establishing links between serum thyroid hormone panel and triglyceride (TG) concentrations with non-invasively obtained measurements of anthropometric parameters of young women may provide preliminary knowledge about the homeostasis of metabolic processes and body composition and about the strategic role of the tested parameters as early screening tests for assessing the health status of apparently healthy women in the period preceding pregnancy. The study was conducted in 381 healthy female students (aged 18-26 years, mean ± SD = 22.1 ± 1.3). Anthropometric indices (BMI, waist-to-hip ratio, FAT%) were calculated and serum concentrations of thyroid hormones (TSH, fT3, fT4) were determined using electrochemiluminescence immunoassays and serum triglycerides (TG) with a commercially available test. No association was established between serum TSH and anthropometric indices in healthy young women. Increased serum concentrations of fT4, fT3 and TG were found in overweight subjects, i.e. BMI > 24.9 kg/m2 (p < 0.05). A significant negative association between fT3 and TG was found in underweight subjects (r = - 0.258, p = 0.049) and a significantly positive association in normal-weight subjects (r = 0.139, p = 0.019). In healthy young women differences in BMI are not related to thyroid function. The opposite directions between the associations fT3 vs TG in underweight compared to normal-weight young prepregnant females may suggest dependencies of fT3 and TG in the regulation of specific BMI-dependent metabolic processes.

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.

Serum indoxyl sulphate and its relation to albumin and α1-acid glycoprotein as a potential biomarkers of maternal intestinal metabolism during pregnancy and postpartum.

BACKGROUND: Serum indoxyl sulfate (IS) levels depend on the production of indole in the gut. The biological effects of IS in the vascular bed could be confirmed by changes in the levels of individual serum proteins during normal pregnancy and in the postpartum period as compared with non-pregnant controls. Albumin (Alb) and α1-acid glycoprotein (AGP, orosomucoid) are the most abundant serum carrier proteins with potential interrelationships with serum levels of IS. METHODS: Serum levels of IS, Alb and AGP were measured in 84 pregnant women in the first, second and third trimester of pregnancy and in the postpartum period, as well as in non-pregnant controls (n = 20), using ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry (IS), colorimetric assay (Alb) and immunoturbidimetric assay (AGP). RESULTS: The postpartum serum levels [mg/L] of IS were lower (p = 0.027) than in the second trimester (mean±SD: 0.85±0.39 vs 0.58±0.32). There were no differences in the IS to ALB ratio calculated in the three trimesters of pregnancy, the postpartum period, and in the non-pregnant controls. The IS/AGP ratio increased from the first to the second trimester (p = 0.039), and decreased in the postpartum period (p<0.05), when it was lower than in the second and third trimester. CONCLUSIONS: The variability of the serum IS/AGP ratio during pregnancy and in the postpartum period may reflect shared involvement in the regulation of their intravascular relationships. The link between serum levels of IS derived from the gut and AGP could serve a potential biomarkers of maternal intestinal metabolism during pregnancy and postpartum.

Associations between the thyroid panel and serum protein concentrations across pregnancy.

Establishing any characteristic associations between the serum parameters of thyroid function and serum proteins in pregnancy may aid in elucidating the role of the thyroid gland in the regulation of pregnancy-specific metabolic processes and in selecting candidate biomarkers for use in their clinical assessment. Concentrations of thyroid stimulating hormone (TSH), free tri-iodothyronine (fT3) and free thyroxine (fT4), six electrophoretically separated protein fractions (albumin, alpha-1-, alpha2-, beta-1-, beta-2- and gamma-globulins), representative proteins-albumin (ALB), transferrin (TRF), alpha-2-macroglobulin (AMG) and ceruloplasmin (CER) were measured in 136 serum samples from 65 women in their consecutive trimesters of pregnancy. The concentrations of TSH, fT4 and fT3 were significantly correlated (p < 0.05) with the concentrations of the albumin, alpha-2- and beta-1 globulin fractions. Significant correlations (p < 0.05) which were positive between fT4 and ALB and negative between fT4 and TRF were established throughout pregnancy. Significant negative correlations (p < 0.05) were demonstrated for fT3 with alpha-2-globulin, AMG and CER. Changes in the serum concentrations of thyroid hormones seen between the trimesters were found to correlate with the concentrations of high-abundance serum proteins. Opposite directions of correlations between fT4 and ALB and fT4 and TRF observed throughout pregnancy may indicate the shared biological role of these parameters in maintaining maternal homeostasis and they suggest their potential use in the clinic as a simple biomarker panel. A negative correlation of fT3 with CER in the second trimester possibly reflects their involvement in the active regulation of metabolic processes.

Can variability of serum electrophoretic fractions during pregnancy provide knowledge about maternal and fetal health.

AIM: Characteristics of variability of concentrations total protein and its electrophoretic fractions in serum of healthy pregnant women between successive trimesters and post-partum for initial classification of proteins involved in specific metabolic processes associated with pregnancy. METHODS: Total serum protein concentrations were measured by biuret method and serum protein fractions were electrophoretically separated in 166 serum samples collected from healthy pregnant women in three trimesters of pregnancy (1st, n = 55; 2nd, n = 42; 3rd, n = 39) and in post-partum (n = 30), and in 20 samples from nonpregnant controls. RESULTS: Across pregnancy, there were gradual, but occurring at different rates, decreases over time in serum total protein, albumin and gamma globulins compared to controls (P < 0.05). In 1st trimester, serum concentrations of total protein, albumin and gamma globulins were <10% lower than in nonpregnant state, with further decreases in 2nd and 3rd trimesters and in post-partum. The concentrations of alpha-1-, alpha-2-, beta-1- and beta-2-globulins were elevated compared to controls (P < 0.05) with different dynamics of change and with the highest percentage increase for alpha-1-globulin. CONCLUSION: Pregnancy-associated alterations in the serum concentrations of total protein and in its individual electrophoretic protein fractions in each trimester of pregnancy and differences versus normal ranges in nonpregnant healthy females could be a simple screening method for classification useful laboratory parameters that help obstetricians and gynecologists to make multidirectional judgments about the state of health of pregnant women.

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.

Vitamin D-binding protein as a biomarker to confirm specific clinical diagnoses.

Introduction: Vitamin D-binding protein (DBP) performs a variety of functions as a transporter for various ligands and takes part in a number of systemic and local physiological and pathological processes. The knowledge about the pathomechanisms of this protein involvement justifies its use as a biomarker to confirm specific clinical diagnoses suggested by nonspecific signs and symptoms.Areas covered: DBP has properties of both systemic laboratory parameters measured in the blood plasma and specific parameters measured in variety of physiological fluids to assess local changes in specific body organs. Articles published in English between 1993 and 2019 were searched for in PubMed using terms DBP, vitamin D, and metabolites, inflammation. DBP is a transport protein and a regulator of immune and inflammatory processes.Expert opinion: DBP capacity for transporting numerous ligands and co-involvement of DBP in immune and inflammatory processes suggest that DBP may be used in laboratory diagnostics as a specific parameter to confirm pathomechanisms of several systemic diseases and local conditions. Changes in the concentration of DBP present in a variety of clinical material may provide valuable information for use in assessing the severity and treatment of pathological processes.

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.

Protein and peptide profiles in neonatal meconium.

AIM: The proteins accumulated in the meconium reflect the intrauterine environment and are naturally excreted by a neonate. The identification and classification of individual meconium proteins may be a valuable source of information about physiological and pathological processes in utero. METHODS: Proteomic analysis was used to study the protein composition in pooled 50 serial meconium portions from 10 neonates. The proteins were classified based on the gene ontology database. The amounts and relative number of proteins (%) in the identified categories and their subcategories were assessed. RESULTS: A total of 946 proteins identified in meconium, including 430 represented by two or more peptides were classified into three categories: biological process (n = 401), molecular function (n = 386) and cellular component (n = 422). The highest number of proteins (>25% of the total) was found in the subcategories: developmental processes, signaling, transport, response to stimulus, regulation, metabolic processes, ion binding, extracellular region, membrane and cytoplasm. CONCLUSION: The composition of meconium proteins identified in this study may be a rich source of new biomarkers for use in neonatology with a potential to predict later development.

Relationships between meconium concentrations of acute phase proteins.

Meconium concentrations of naturally accumulated ceruloplasmin (CER), lactoferrin (LF), and neutrophil gelatinase-associated lipocalin (NGAL) and their relationships considered as a panel of acute phase proteins could be used for the assessment of fetal homeostasis. CER, LF and NGAL concentrations were measured using enzyme-linked immunosorbent assay kits in meconium portions (n = 80) collected from 19 healthy neonates. The coefficients of variation (CV) of the meconium LF (1.77) and NGAL (1.26) were about two-fold higher than that of CER (0.73) with significant (P < 0.05) correlations between all three parameters. The LF to NGAL ratio (CV = 0.67) correlated strongly with the CER concentrations (r = 0.39, P < 0.01). These correlations between CER, LF and NGAL concentrations suggest their combined involvement in the metabolic processes in the developing fetus.

Variations in serum concentrations of C-reactive protein, ceruloplasmin, lactoferrin and myeloperoxidase and their interactions during normal human pregnancy and postpartum period.

BACKGROUND: Serum proteins may provide information about homeostasis of redox status and inflammatory processes also during pregnancy. The aim of the study was to assess the dynamics of changes in serum concentrations of C-reactive protein (CRP), ceruloplasmin (CP), lactoferrin (LF) and myeloperoxidase (MPO) and their interactions during normal pregnancy and the postpartum period. METHODS: The concentrations of proteins were measured in serum (n=113) from pregnant in consecutive trimesters and in postpartum period (n=28) and in non-pregnant women (n=17), using immunoturbidimetric assays (CRP, CP) and ELISA Kits (LF, MPO). RESULTS: The concentrations [mg/dl] CP and CRP (mean±SD respectively): second trimester (43.1±6.2; 0.49±0.57), third trimester (44.5±5.8; 0.41±0.37), postpartum (42.39±6.4; 4.15±3.6) were higher than in the first trimester (33.0.5±8.7; 0.31±0.36) or non-pregnant women (24.12±7.4; 0.12±0.13). The increases in concentrations of CP and CRP between the first and the second trimesters were by approximately 35% and 50% respectively and the correlation coefficients in the first trimester and in non-pregnant women were twice higher than in the second trimester and the postpartum period. The concentrations [µg/ml] LF and MPO were no significant differences (mean±SD respectively): first (6.19±4.54; 0.17±0.12), second (5.68±4.4; 0.14±0.08), third (6.34±6.98; 0.17±0.14), the postpartum (4.86±3.64; 0.25±0.4), and non-pregnant (3.9±2.56; 3.2; 0.14±0.05). However, significant correlations were established (p<0.05) between MPO and LF in all groups and between the following ratios CRP/LF vs CP/MPO and CRP/MPO vs CP/LF. CONCLUSIONS: The concentrations of proteins synthesized by the liver (CP, CRP) dynamically increase during consecutive trimesters of pregnancy unlike neutrophil-derived proteins (LF, MPO). Statistically significant correlations between the proportions of the serum proteins may suggest their combined role for the maintenance of homeostasis during pregnancy.

Calprotectin in Serially Collected Meconium Portions as a Biomarker for Intrauterine Fetal Environment.

OBJECTIVE: Understanding the pathomechanisms underlying high meconium calprotectin concentrations is the key to the potential uses of this parameter for the assessment of the intrauterine environment in which the fetus develops. The aim of this study was to measure calprotectin concentrations in serial meconium portions passed after birth and to calculate the individual variations in the total meconium calprotectin content accumulated during gestation. METHODS: Calprotectin concentrations were measured using Calprotectin ELISA kit (Immundiagnostik AG) in all meconium portions (n = 81) from 20 healthy neonates. For each neonate, total meconium calprotectin was calculated, reflecting the sum of calprotectin content in all meconium portions from this neonate. RESULTS: The calprotectin concentration in meconium was (mean ± SD) 286.5 ± 214.6 µg/g (range 34.7-1,067.1). Calprotectin concentrations in the last portions passed were nearly 3-fold higher than in the first portions (p = 0.0004). The total individual calprotectin content of (mean ± SD) 3,668.7 ± 1,819.0 µg (range 1,158.9-8,155.5) was related to the birth weight (r = 0.46, p = 0.042). CONCLUSIONS: Wide intra- and interindividual differences in calprotectin concentrations in the meconium may reflect intestinal inflammation associated with the fetal adaptation to life outside the uterus. Calprotectin may serve as a biomarker useful for the identification of endogenous and exogenous factors with impact on the intrauterine environment.

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.

Meconium lactoferrin levels in neonates: can we predefine normal values?

BACKGROUND AND AIMS: In recent years, Lactoferrin (LF) has become an object of interest to neonatologists. To date, there have been no studies on the presence of LF in neonatal meconium. The aim of the study was to assess LF concentrations in successive portions of meconium passed in the first days of extrauterine life and to calculate the total amount of LF accumulated in the fetal intestine in utero. METHODS: The LF concentrations were determined using the ELISA Kit in meconium samples (n = 81), collected serially from neonates (n = 20). The sum of LF amounts in all portions of meconium passed by a neonate was considered to represent the total accumulation of this protein in the fetal intestine in utero. RESULTS: The LF concentration in a single meconium portion was [µg/g]: mean ± SD = 45.07 ± 78.53, median = 18.98, range = 1.69-511.43. The total LF accumulation in the fetal intestine was [µg]: mean ± SD = 757.23 ± 745.41, median = 514.73, range = 20.48-2749.55. LF concentrations increased in the last meconium portions compared with the first portions passed immediately after birth (p = 0.017). CONCLUSIONS: Very large differences in LF concentrations between meconium portions and in the total LF accumulation between the neonates suggest the influence of intrauterine factors on the variations in fetal intestinal LF concentrations.