Barbara Lisowska-Myjak

Serum and Urinary Biomarkers of Acute Kidney Injury

Acute kidney injury (AKI) is a frequent clinical problem in critically ill patients and the associated mortality is high. Standard serum and urine biomarkers are insensitive and nonspecific for the detection of kidney injury in its early stages which limits the therapeutic options and may compromise the outcome. The study presents new candidates for biochemical markers of AKI, with potentially high sensitivity and specificity, causally related to its pathogenesis and development. Some of these biomarkers measured in serum or urine are well known in laboratory practice but have been used in other tests, while some novel biomarkers have been proposed as a result of experimental and clinical studies. In current clinical practice, identification and classification of AKI is based on elevations in serial serum creatinine concentrations, which are delayed and therefore unreliable in the acute setting. The most promising of the new serum AKI markers are cystatin C, neutrophil gelatinase-associated lipocalin and uric acid. Urinary AKI markers may be classified as enzymes released from damaged tubular cells (alkaline phosphatase, γ-glutamyl transpeptidase, alanine aminopeptidase, isoenzymes of glutathione transferase, N-acetyl-β-D-glucosaminidase), low-molecular-weight proteins (α1-microglobulin, β2-microglobulin, retinol-binding protein, cystatin C) and proteins specifically produced in the kidney and associated with the development of AKI [cysteine-rich protein 61, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, cytokines and chemokines (Gro-α, IL-18), and structural and functional proteins of renal tubules (F-actin, Na+/H+ exchange isoform 3)]. Based on the different expression of these markers, using a panel of serum and urine markers may potentially help to distinguish between various types of insults, establish the duration and severity of injury, predict the clinical outcome and help to monitor response to treatment in AKI.

Serum alpha-1-antitrypsin concentration during normal and diabetic pregnancy

OBJECTIVES The aim of this study was a comparison of serum alpha-1-antitrypsin (AAT) concentration in the course of normal and diabetic pregnancy. METHODS Serum AAT concentration was determined on NOR-Partigen plates (Behring Diagnostics GmbH, Marburg). The studied material included healthy women without pregnancy (n=14), healthy pregnant women in the first trimester (n=12), second trimester (n=15), third trimester (n=15), and 16 pregnant women with type-1 diabetes mellitus studied prospectively in successive stages of pregnancy. RESULTS In the first trimester of normal pregnancy, a significant increase of serum AAT concentration was observed in comparison with healthy women without pregnancy (P<0.01). In all stages of pregnancy with type-1 diabetes mellitus, a higher increase of AAT concentration was found as compared with healthy pregnant women (P<0.0001), especially in third trimester. There was no correlation shown between concentration of AAT and fructosamine in the serum of healthy and diabetic pregnant women (P>0.05). CONCLUSIONS During normal and diabetic pregnancy, an increase of serum AAT concentration occurred with the regression lines exhibiting a different slopes. The highest AAT concentration was observed in third trimester of diabetic pregnancy. Increase in concentration of AAT in the serum of pregnant women with diabetes does not depend on the value of glycaemic control.

Antigenic and functional levels of alpha-1-antitrypsin in serum during normal and diabetic pregnancy

OBJECTIVES Serum alpha-1-antitrypsin (AAT) concentration may not be representative of the functional capacity of this inhibitor. The aim of this study was to determine the antigenic and functional serum levels of AAT during normal and diabetic pregnancy. METHODS Serum AAT concentration was measured on NOR-Partigen plates (Dade Behring). Trypsin inhibitory capacity (TIC) in the serum was determined with N-benzoyl-DL-arginine-p-nitroaniline (BAPNA, Sigma) as substrate. The examined material included pregnant women with type 1 diabetes mellitus (n=16) studied prospectively in successive stages of pregnancy, healthy pregnant women in the first trimester (n=12), second trimester (n=15), third trimester (n=15) and healthy non-pregnant women (n=14). RESULTS Serum concentration of AAT in all consecutive phases of diabetic pregnancy is higher as compared to normal pregnancy (P<0.0001). Serum TIC is significantly lower in the first and third trimesters in diabetic pregnancy (P<0.05, 0.001, respectively). Specific activity of serum AAT (mg of trypsin inhibited by 1mg of AAT) does not change between subsequent trimesters both in normal and diabetic pregnancy and in diabetic pregnancy is two times lower as compared to normal pregnancy. CONCLUSION In spite of the higher level of AAT in the serum in diabetic pregnancy, the ability of this inhibitor to inhibit trypsin is two times lower as compared to normal pregnancy.

Urinary proteins, N‐acetyl‐β‐D‐glucosaminidase activity and estimated glomerular filtration rate in hypertensive patients with normoalbuminuria and microalbuminuria

Background:  The aim of the study was to assess novel candidate markers measured in the urine of normoalbuminuric and microalbuminuric patients (the urinary albumin‐to‐creatinine ratio < 30 mg/mmol) with essential hypertension to be used for early detection and assessment of progressive deterioration in renal function.

Alpha-1-antitrypsin and IgA in serial meconium and faeces of healthy breast-fed newborns.

Background: Meconium is a series of layers formed in the foetal intestine from the 12th week of gestation. High content of meconial alpha-1-antitrypsin (AAT), decreasing within the first several days of extrauterine life appears to reflect the meconium clearance of the gut. At birth, IgA is not present in the meconium and breast-fed infants receive this antibody postnatally with human milk. The aim of the study was to determine changes in AAT concentrations, functional activity of that inhibitor expressed as trypsin inhibitory capacity (TIC) and IgA concentration in serial meconium and faeces, as endogenous biochemical markers discriminating between faeces portions formed in intrauterine and extrauterine life periods of healthy breast-fed newborns. Methods: A group of 24 healthy breast-fed newborns delivered by spontaneous labour were studied prospectively during the first 4 days of postnatal life. AAT and IgA concentrations in the newborn‘s meconial and faecal samples and IgA concentration in mother’s milk samples taken on the third day after delivery, were determined by radial immunodiffusion. TIC was assessed using BAPNA (N-benzoyl-DL-arginine-p-nitroanilide). Results: The medians (range) of AAT concentrations in milligrams per gram of dry meconium or faeces were: 68.8(29.2–138.4) (day 1), 56.9 (30.8–112.8) (day 2), 26.2 (6.8–80.7) (day 3), and 6.6 (1.4–27.1) (day 4). The medians (range) of TIC in milligrams of trypsin/g dry mass of meconium or faeces were: 0.76 (0.33–1.79) (day 1), 0.44 (0.17–1.08) (day 2), 0.16 (0.03–0.56) (day 3), and 0.03 (0–0.11) (day 4). The median (range) of IgA concentration in mothers’ milk was 715 mg/dl (420–890). IgA was absent in meconium portions from the first day of life while on the successive days the medians (range) of IgA concentration in mg/g dry mass of meconium and faeces were as follows: 0 (0–2.90) (day 2), 2.50 (1.10–9.60) (day 3), 7.05 (4.10–30.60) (day 4). On day 4 of extrauterine life a negative correlation was found between AAT and IgA concentrations in faeces of the newborns (r = –0.46) and a positive correlation was seen between IgA concentrations in faeces and milk (r = 0.93). Conclusions: Analyses of the systematic decrease in AAT and increase of IgA concentration in serial portions of meconium and faeces over the first days of extrauterine life of breast-fed newborns can date newborn’s faeces portions formed during intrauterine and extrauterine maturation. AAT deposited in foetal intestine is an active antiprotease.

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-α-tosyl-l-arginine-p-nitroanilide) as substrate, trypsin inhibitory capacity (TIC) with N-α-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 newborns 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 newborns pancreatic exocrine functions.

Concentrations of neutrophil-derived proteins in meconium and their correlations

AIM The aim was to measure concentrations of four neutrophil-derived proteins in meconium as biomarkers describing prenatal environment. METHODS Calprotectin, lactoferrin, myeloperoxidase and PMN-elastase concentrations were measured using ELISA kits in serial meconium portions (n = 81) from 20 healthy neonates. RESULTS The highest concentration was for calprotectin (286.5 ± 214.6 µg/g) with a positive correlation (r = 0.75, p < 0.0001) with myeloperoxidase (1.81 ± 1.72 µg/g). For PMN-elastase (1.70 ± 2.69 µg/g) a negative correlation was observed with calprotectin and myeloperoxidase (r = -0.51, p < 0.0001; r = -0.60, p < 0.0001, respectively). Concentration of lactoferrin (45.07 ± 78.53 µg/g) correlated only with that of myeloperoxidese (r = 0.36, p = 0.0009). CONCLUSION Calprotectin, lactoferrin, myeloperoxidase and PMN-elastase concentrations in meconium are interrelated. These proteins may serve as objective biomarkers describing and/or assessing the intrauterine environment.

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.

Role of Uremic Compounds in Organ Injury

All substances with molecular weights up to 58 kDa retained in the blood as the results of renal dysfunction are potential uremic toxins. The search for endogenous toxic compounds seems to offer a novel approach to identifying and explaining any so far unexplored specific effects on the body organs and systems. In contemporary laboratory diagnostics there are no suitable markers for use in comprehensive evaluation of complex toxicity of uremic compounds accumulated in successive stages of developing renal dysfunction. To provide a sound basis for treatments which would effectively protect against or slow down multiple organ injury caused by uremic toxins novel parameters are needed, more specific than urea and creatinine. Identification of reliable biomarkers or their panels needs careful consideration of their concentrations in biological materials, biological activity and usefulness for effective diagnosis. Classification of uremic compounds, based on their chemical properties, role in pathophysiological processes and the organs where they are formed remains to be elucidated with meticulous observation of clearly formulated rules guiding the process.

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

Abstract 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.