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.

Degradation of Lung Protective Angiotensin Converting Enzyme-2 by Meconium in Human Alveolar Epithelial Cells: A Potential Pathogenic Mechanism in Meconium Aspiration Syndrome.

BACKGROUND: Pancreatic digestive enzymes present in meconium might be responsible for meconium-induced lung injury. The local Renin Angiotensin System plays an important role in lung injury and inflammation. Particularly, angiotensin converting enzyme-2 (ACE-2) has been identified as a protective lung enzyme against the insult. ACE-2 converts pro-apoptotic Angiotensin II to anti-apoptotic Angiotensin 1-7. However, the effect of meconium on ACE-2 has never been studied before. OBJECTIVE: To study the effect of meconium on ACE-2, and whether inhibition of proteolytic enzymes present in the meconium reverses its effects on ACE-2. METHODS: Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium, meconium + a protease inhibitor cocktail (PIc) and PIc alone for 16 h. At the end of incubation, apoptosis was measured with a nuclear fragmentation assay and cell lysates were collected for ACE-2 immunoblotting and enzyme activity. RESULTS: Meconium caused a fourfold increase in apoptotic nuclei (p < 0.001). The pro-apoptotic effect of meconium can be reversed by PIc. Meconium reduced ACE-2 enzyme activity by cleaving ACE-2 into a fragment detected at ~ 37 kDa by immunoblot. PIc prevented the degradation of ACE-2 and restored 50% of ACE-2 activity (p < 0.05). CONCLUSION: These data suggest that meconium causes degradation of lung protective ACE-2 by proteolytic enzymes present in meconium, since the effects of meconium can be reversed by PIc.

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.

Correlations between ceruloplasmin, lactoferrin and myeloperoxidase in meconium.

BACKGROUND AND AIMS: Oxidative stress and the generation of reactive oxygen/nitrogen species has a known significant impact on intrauterine fetal growth and the risk of metabolic diseases in adulthood. Compounds accumulated in fetal meconium may be a source of information about the oxidoreductive status during the intrauterine development. Three metal-containing proteins ceruloplasmin (CP), lactoferrin (LF) and myeloperoxidase (MPO) constitute the complementary panel modulating oxidative stress. The aim of this study was to assess the concentrations of these proteins and their correlations in meconium from healthy neonates. METHODS: The CP, LF and MPO concentrations were determined using ELISA Kits. All serial meconium portions (n=80) were collected from healthy full-term neonates (n=19). RESULTS: The mean±SD concentrations [µg/g] in meconium samples were as follows: CP 312.4±229.7 (range 52.2-1076), LF 45.6±78.9 (range 1.7-511.4), MPO 1.8±1.7 (range 0.02-8.8) with statistically significant correlations between CP vs. LF (R=0.459, p=0.00009) and LF vs. MPO (R=0.354, p=0.0013). A statistically significant increase in the concentrations (p<0.05) between the first and the last meconium portions was found for LF (p=0.027) and for MPO (p=0.0006). CONCLUSIONS: Strong correlations between the meconium concentrations of CP, LF and MPO indicate a possible role of these complementary proteins in maintaining homeostasis of the intrauterine environment of the fetus. CP, LF and MPO measured in meconium may serve as biomarkers for assessment of impairment of oxidative balance during intrauterine life with its potential impact on disease development in adulthood.

A new look at the pathogenesis of the meconium aspiration syndrome: a role for fetal pancreatic proteolytic enzymes in epithelial cell detachment.

We hypothesized that fetal pancreatic digestive enzymes play a role in the lung damage after meconium aspiration. We studied the effect of meconium on the A549 alveolar epithelial cell line. The exposure of the cells to 0.5 to 5% meconium resulted in significant disruption of connections between A549 cells and caused dose-dependent cell detachment, without signs of cell death. A protease inhibitor cocktail prevented the A549 cell detachment induced by meconium. After the exposure to 2.5% meconium, a protective effect was quantified by measuring light absorbance by gentian violet stain of still attached cells. The protease inhibitor cocktail and chymostatin showed significant protective effects, increasing the number of attached cells by 135 and 123%, respectively (p < 0.05). Other individual protease inhibitors tested in the detachment assay (AEBSF, leupeptin, E-64, aprotinin, benzamidine, phosphamidon, and aminohexanoic acid) did not offer statistically significant protection. These results afford a new perspective on the pathophysiology of meconium aspiration syndrome (MAS). We speculate that disruption of intercellular connections and cell detachment from the basement membrane are key events in the pathology associated with MAS. The observed protective effects of protease inhibitors suggest that they may be useful in the treatment and/or prophylaxis of MAS.

Human meconium contains significant amounts of alkaline sphingomyelinase, neutral ceramidase, and sphingolipid metabolites.

Intestinal alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase may catalyze the hydrolysis of endogenous sphingomyelin (SM) and milk SM in human-milk fed infants. The enzymes generate sphingolipid metabolites that may influence gut maturation. Alk-SMase also inactivates platelet-activating factor (PAF) that is involved in the pathogenesis of necrotizing enterocolitis (NEC). We examined whether the two enzymes are expressed in both preterm and term infants and analyzed Alk-SMase, neutral ceramidase, SM, and sphingolipid metabolites in meconium. Meconium was collected from 46 preterm (gestational ages 23-36 wk) and 38 term infants (gestational ages 37-42 wk) and analyzed for Alk-SMase using C-choline-labeled SM and for neutral ceramidase using C-octanoyl-sphingosine as substrates. Molecular species of SM, ceramide, and sphingosine were analyzed by high-performance liquid chromatography mass spectroscopy. Meconium contained significant levels of Alk-SMase and ceramidase at all gestational ages. It also contained 16-24 carbon molecular species of SM, palmitoyl- and stearoyl-sphingosine, and sphingosine. There were positive correlations between levels of SM and ceramide and between ceramide and sphingosine levels. In conclusion, Alk-SMase and ceramidase are expressed in the gut of both preterm and term newborn infants and may generate bioactive sphingolipid messengers.

Trypsin and antitrypsin activities and protein concentration in serial meconium and feces of healthy newborns.

BACKGROUND: Meconium has the potential for being the matrix on which markers of fetal exposure to physiologic and non-physiologic agents during intrauterine life can be analyzed. The aim of this study was to compare trypsin and antitrypsin activities and protein concentration during intra- and extrauterine human development based on an assessment of these parameters in serial meconium and first feces of healthy, term newborns during the first four days of life. METHODS: One hundred and eighteen mature, term newborns were studied. Single portions of meconium or feces were taken prospectively from day 1 to day 4. In ten newborns each meconium and feces passed by the infant from birth up to the fourth day after delivery was collected individually. Trypsin activity was measured using L-TAPA (N-alpha-tosyl-l-arginine-p-nitroanilide) as substrate, trypsin inhibitory capacity (TIC) with N-alpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate, and protein concentration by the method of Lowry et al. RESULTS: Meconium and feces of healthy newborns demonstrated a decrease in TIC during the first two days of life and an increase in trypsin activity over the first three successive days of life. No correlation was observed between the variability of both parameters. During the first three days of a newborn's life, significant correlation was observed between TIC and protein concentration in meconium and first feces. In the course of proteolytic activity changes in successive portions of meconium and feces, a transient peak occurs on the 2nd-4th day of life. CONCLUSIONS: The gradual decrease in protease inhibitor activity and low trypsin activity in successive portions of meconium from the first two days of extrauterine life may provide a retrograde chronological depiction of the course of the second and third trimesters of intrauterine life. Serial collection of feces over 2-4 days may provide a reflection of the dynamics of the postnatal increase in the newborn's pancreatic exocrine functions.

Faecal elastase 1 levels in premature and full term infants.

BACKGROUND: Determination of faecal elastase 1 (FE1) is a simple, relatively inexpensive, non-invasive, highly specific and sensitive test for determining pancreatic function. Secretion of pancreatic enzymes varies during infancy, but there are almost no specific data on the ontogeny of elastase 1 in human babies. AIM: To study FE1 levels in preterm and term babies, and to determine the possible effect of gestational and postconceptual age on these levels. METHODS: Serial stool samples were collected and tested for FE1 level from 77 premature and full term infants. FE1 levels were determined by a commercially available enzyme linked immunosorbent assay (ELISA) kit. RESULTS: A total of 232 stool samples were collected from 77 neonates. The FE1 level measured in the first stool sample (meconium) was below normal (200 micro g/g stool) in all samples regardless of gestational age. Sixty three neonates had at least two samples tested for FE1 level. The mean (SD) level of FE1 in sample 1 was 45.9 (51.1) micro g/g stool and was significantly (p < 0.001) lower than in sample 2 (243.0 (164.9) micro g/g stool). The lower the gestational age of the newborn, the more time it took for FE1 to reach normal levels. CONCLUSIONS: FE1 levels in meconium are low, and studies in meconium should be avoided if pancreatic sufficiency is to be determined. FE1 reaches normal levels by day 3 in term newborns and by 2 weeks in infants born before 28 weeks gestation. Normal levels are reached sooner in infants of more advanced gestational age who start enteral feeding earlier.

Phospholipase A2 is present in meconium and inhibits the activity of pulmonary surfactant: an in vitro study.

UNLABELLED: Atelectasis, a major contributor to pulmonary dysfunction in meconium aspiration syndrome (MAS), is produced by bronchiolar obstruction and surfactant inactivation. It has been shown that substances in meconium, e.g. fatty acids, inhibit surfactant activity. However, the role of the enzyme phospholipase A2 (PLA2), which hydrolyses surfactant in adult respiratory distress syndrome (ARDS), has not yet been studied. Our objective was to investigate whether PLA2 is present in meconium and inhibits pulmonary surfactant activity in vitro. Therefore, the presence of PLA2 activity in meconium, collected from 10 newborns, was measured by the formation of lysophosphatidylcholine after incubation of meconium with radioactively labelled dipalmitoylphosphatidylcholine. Meconium was fractionated by Sephadex G-100 column chromatography and the fractions were assayed for PLA2 activity. Also, their effect on the surface tension of surfactant (Curosurf) was measured using a pulsating bubble surfactometer (PBS). PLA2 activity was present in all meconium samples. Addition of meconium to surfactant significantly increased surface tension (mean +/- SD: 1.7 +/- 1.6 mN/m to 24.3 +/- 6.7 mN/m, p = 0.0001) and only the addition of the PLA2 containing fraction from meconium to surfactant also significantly increased surface tension (mean 1.7 +/- 1.6 mN/m to 19.0 +/- 3.58 mN/m, p < 0.0001). CONCLUSION: PLA2 is present in meconium and inhibits the activity of pulmonary surfactant in vitro. Therefore, PLA2 in meconium may contribute to surfactant inactivation and alveolar atelectasis in MAS.

Human meconium has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet lungs.

Aspiration of meconium produces an inflammatory reaction resulting in necrotic changes in lung tissue. To further investigate the mechanisms of the meconium-induced early pulmonary injury, twenty 10-12-d-old piglets were studied for lung tissue ultrastructural and apoptotic changes and phospholipase A2 activity. Twelve piglets received an intratracheal bolus (3 mL/kg) of a 20-mg/mL (thin, n = 6) or 65-mg/mL (thick, n = 6) mixture of human meconium, and control piglets (n = 5) received the same amount of intratracheal saline. Three ventilated piglets with no aspiration were also studied. Pulmonary hemodynamics and systemic oxygenation were followed for 6 h after meconium or saline insufflation. In the control groups, the pulmonary tissue showed open alveolar spaces and intact vascular walls, whereas meconium administration resulted in severe pneumonitis, with alveolar spaces filled with inflammatory exudate. Meconium instillation additionally resulted in edematous changes in the vascular walls and alveolar epithelium, whereas type II pneumocytes were intact. The amount of apoptotic cells was increased, especially in the respiratory epithelium, and the catalytic activity of phospholipase A2 in lung tissue samples was significantly elevated after thick meconium instillation. This activity rise proved to be mainly because of human group I phospholipase A2, introduced by meconium. Our data thus show that aspiration of meconium leads to severe lung tissue inflammation with early ultrastructural changes in the pulmonary alveolar walls and is associated with apoptotic cell death in the epithelium, already during the first hours after the insult. These results further suggest that high phospholipase A2 activity, mainly introduced into the lungs within the meconium, may have an important role in the initiation of these alterations in neonatal lungs.

Inhibition of beta-glucuronidase by casein hydrolysate formula.

BACKGROUND: A casein hydrolysate infant formula has been shown to be associated with lower levels of neonatal jaundice than are standard infant formulas. Because beta-glucuronidase is related to neonatal jaundice, this study examined the effect of a casein hydrolysate formula on beta-glucuronidase. METHODS: Beta-glucuronidase activity was measured with or without added dietary components. The beta-glucuronidase sources used were meconium, breast milk, and the purified bovine liver enzyme. The dietary components assayed for their effect on beta-glucuronidase activity included casein hydrolysate formula (Nutramigen), whey-predominant formula (Enfamil), breast milk; enzymatically hydrolyzed casein, and other constituents of the casein hydrolysate formula. Stool samples of 6-day-old infants, who were exclusively fed one of the two formulas or breast milk, were also assayed for inhibition of beta-glucuronidase. RESULTS: Only Nutramigen, enzymatically hydrolyzed casein, and stool from Nutramigen-fed infants consistently demonstrated significant inhibition of beta-glucuronidase activity, ranging from 45% to 85% of that in controls. The inhibition of beta-glucuronidase in purified bovine liver demonstrates a dose response in a pH range from 4 to 7.3. CONCLUSIONS: Hydrolyzed casein contains a beta-glucuronidase inhibitor that, in casein hydrolysate-fed infants, persists after passage through the digestive tract. These data are consistent with the possibility that inhibition of beta-glucuronidase is a mechanism by which infants fed casein hydrolysate have lower jaundice levels than infants fed routine formulas or breast milk. Further study of this mechanism is needed.

Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells.

A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.

[Proteolytic enzymes in meconium--studies of healthy newborn infants and premature infants]. / Proteolytische Enzyme des Mekoniums--Untersuchungen bei gesunden Neugeborenen und bei Frühgeborenen.

Peptidatic activity of meconium of 200 newborns and 100 preterm babies. Protein content of meconium is higher in preterm babies than in healthy newborns referred to fresh weight. Enzymatic activity opposite to Gly-D-Leu is higher in term than in preterm newborns. It is on the contrary in the case of alanine-aminopeptidase, dipeptidyl-peptidase IV and the activities opposite to Leu-NA and Arg-NA. The good correlated enzymatic activities opposite the different substances give a hint to the presence of alanine-amino-peptidase (EC 3.4.11.2.)

Chemical nature of intestinal-type alkaline phosphatase in human kidney.

Approximately 10% of the alkaline phosphatase activity in human kidney is derived from the intestinal-type alkaline phosphatase isoform, which can be differentiated from adult intestinal alkaline phosphatase by selective reactivity with monoclonal antibodies. The NH2-terminal sequence of the renal intestinal-type alkaline phosphatase was shown to be identical to sequences of the adult and meconial alkaline phosphatases except for the NH2-terminal valine residue, which is missing in the renal intestinal-type enzyme. Incubation of purified meconial alkaline phosphatase with kidney homogenate resulted in removal of the NH2-terminal valine residue, indicating the presence of aminopeptidases in kidney that catalyze this hydrolysis. Furthermore, the oligosaccharide chains of the renal intestinal-type alkaline phosphatase were shown to differ from those of meconial and adult intestinal alkaline phosphatases, as revealed by lectin affinity chromatography. The heterogeneity of the intestinal-type alkaline phosphatase can therefore be generated both by partial peptide bond hydrolysis and differences in glycosylation.

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.

Human fetal intestinal alkaline phosphatase: molecular heterogeneity and immunological detection in amniotic fluids.

Human fetal intestinal alkaline phosphatase (fIALP) is present in amniotic fluids as free dimers (Mr 140,000) or membrane-bound through phosphatidylinositol residues. Extraction of corresponding particulate material with Triton X-100, resulted in release of tetrameric high Mr fIALP forms (Mr 380,000). In individual amniotic fluids, as well as in meconeum, both dimeric and tetrameric fIALP are sialylated to various extents. When measured by a double sandwich-ELISA, up to 10-fold higher fIALP antigen levels were found in amniotic fluids than when determined by an enzyme antigen immunoassay, based upon fIALP enzyme activity measurements. Frequency analysis of fIALP antigen levels, showed a more symmetrical distribution than analysis of fIALP enzyme activities; likewise, the lower 95% confidence limit, calculated for the fIALP antigen distribution curve, overlapped less with the bulk of values. In cystic fibrosis amniotic fluids, measurements of fIALP antigen levels resulted in a lower false-negativity outcome than fIALP enzyme activity measurements, whereas in amniotic fluids of trisomy pregnancies fIALP enzyme activities and fIALP antigen levels were equally unpredictive.

Intestinal-like alkaline phosphatase expressed in normal human adult kidney.

Human adult kidney was found to contain not only the 'tissue-unspecific alkaline phosphatase' but also another alkaline phosphatase isozyme. By use of monoclonal antibodies specific for human intestinal alkaline phosphatase, this kidney isozyme was purified to homogeneity by immunoaffinity chromatography. The structural and kinetic properties of the enzyme were compared with those of the other alkaline phosphatase isozymes expressed in normal human tissues, i.e. the placental, intestinal, meconial (fetal), liver and kidney isozymes. The new kidney isozyme was clearly different from both the tissue-unspecific and the adult intestinal alkaline phosphatase as regards isoelectric point, molecular mass and peptide maps after cyanogen bromide cleavage, but it was found to be identical to the meconial alkaline phosphatase. The results demonstrate simultaneous expression of two alkaline phosphatase isozymes in human kidney, one of which is normally related only to the fetal intestine.

A monoclonal antibody capture assay for intestinal alkaline phosphatase and the measurement of this isoenzyme in pregnancy.

Intestinal ALP was purified from meconium to a specific activity of 1,100 U/mg protein and used as antigen in the preparation of 7 monoclonal antibodies. Two of these antibodies were specific for intestinal ALP and reacted with different epitopes. Both bound adult intestinal ALP better than fetal intestinal ALP. One of these antibodies was used to establish a capture assay for intestinal ALP in human serum over the range 0.5-16 U/l. Reference ranges of serum intestinal ALP concentrations were established in relation to blood groups. Measurement of intestinal ALP in the serum of pregnant women showed no correlation with pre-term fetal passage of meconium.

Protease inhibitor and defective proteolysis in cystic fibrosis.

Meconium specimens from 18 infants with cystic fibrosis (CF) had strong trypsin inhibitory activity (TIA). The same specimen, which contained increased quantities of undigested proteins, had normal concentrations of immunoreactive trypsin (IRT), but deficient trypsin catalytic activity (TCA). TIA was not detected in any specimen from non-CF infants who had high concentration of proteins comparable to that of CF infants. Subjecting meconium supernatant of CF infants to Sephadex G-75 gel filtration revealed that TCA was greatly enhanced in effluents after fractions were activated by porcine trypsin. TCA was present in the same fractions with IRT. The findings suggested that proteases were secreted into the intestinal lumen in CF infants prior to birth. Deficient proteolysis in the disease might be due to the presence of a trypsin inhibitor.